Use of opioid antagonists to attenuate endothelial cell proliferation and migration

ABSTRACT

The invention provides methods of attenuating, e.g., inhibiting or reducing, cellular proliferation and migration, particularly endothelial cell proliferation and migration, including that associated with angiogenesis, using opioid antagonists, including, but not limited to, those that are peripherally restricted antagonists.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/972,122, filed Aug. 21, 2013, which is a continuation of U.S.application Ser. No. 11/379,010, filed Apr. 17, 2006, now U.S. Pat. No.8,524,731 issued Sep. 3, 2013, which is a continuation-in-part of PCTApplication No. PCT/US2006/07892 filed on Mar. 7, 2006, which claimsbenefit under 35 U.S.C. 119(e) of the filing dates of U.S. Ser. No.60/659,193 filed on Mar. 7, 2005, 60/725,703 filed Oct. 12, 2005,60/731,009 filed on Oct. 28, 2005, and 60/760,851 filed Jan. 20, 2006,the entire disclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by National Institutes of Healthgrants: DE12322; DE00470; and DE015830. The United States government hascertain rights in this invention.

FIELD OF INVENTION

The invention relates to methods of attenuating migration and/orproliferation of endothelial cells, especially associated with tumors,utilizing opioid antagonists.

INTRODUCTION

Cellular proliferation is a normal ongoing process in all livingorganisms that involves numerous factors and signals that are delicatelybalanced to maintain regular cellular cycles. Whether or not mammaliancells will grow and divide is determined by a variety of feedbackcontrol mechanism, which includes the availability of space in which acell can grow, and the secretion of specific stimulatory and inhibitoryfactors in the immediate environment.

Angiogenesis and angiogenesis-related diseases are affected by cellularproliferation. The process of angiogenesis results in the formation ofnew blood vessels. Under normal physiological conditions, animals,including humans, undergo angiogenesis only in very specific restrictedsituations. For example, angiogenesis is normally observed in woundhealing, fetal and embryonic development, and formation of the corpusluteum, endometrium and placenta.

During the process of angiogenesis, endothelial cells, which normallyexist in a quiescent state as part of an existing blood vessel, enter amigratory, proliferative state. This migratory, proliferative state ofendothelial cells is eventually resolved when the cells return to thequiescent state as part of a functional new blood vessel. The generationof new capillaries involves a complex process that requires a number ofcellular and molecular events to occur in both a spatial and temporalpattern. Some of these activities include the degradation of thesurrounding basement membrane of the originating vessel, the migrationof the endothelial cells through the connective tissue stroma, cellproliferation, the formation of tube-like structures, and the maturationof these endothelial-lined tubes into new blood vessels. (Cliff, 1963;Schoefl, 1963; Ausprunck and Folkman, 1977). Some essential angiogenicfactors include fibroblast growth factor-basic, vascular endothelialgrowth factor (VEGF), angiopoietins, cytokines, extracellular matrixproteins, and matrix metalloproteases. These factors are producedlocally by stromal cells and by activated leukocytes that are recruitedto the area (Risau, W. (1997) Nature 386(6626):671-674; Risau and Flamme(1995) Ann. Rev. Cell Dev. Biol. 11:73-91). Unlike other angiogenicfactors, VEGF acts as an endothelial cell specific mitogen duringangiogenesis (Terman et al., 1992 and Ferrara, 1993).

Angiogenesis can be stimulated and harnessed by some neoplasms (e.g.,tumors) to increase nutrient uptake. It has been found that angiogenesisis essential for the growth of solid tumors beyond 2-3 mm in diameterand for tumor metastasis (Folkman, 1995; reviewed in Bouck et al.,1996). In contrast to normal angiogenesis, which leads to anastomosesand capillary maturation, angiogenesis associated with neoplasia is acontinuous process. Endothelial cells are activated by nearby neoplasticcells to secrete not only VEGF which stimulates angiogenesis, but alsomatrix metalloproteases (MMP) which degrade the surroundingextracellular matrix. The endothelial cells then invade theextracellular matrix where they migrate, proliferate, and organize toform new blood vessels, which support neoplasm growth and survival. Thenewly vascularized neoplasm continues to grow, leading to furthernutrient deprivation and chronic pro-angiogenic signaling. Thevasculature of neoplasms is characterized by the presence of lacunae anda low rate of anastomoses. This partially dysfunctional vasculaturefuels the permanent requirement for angiogenesis. Additionally, thisincomplete vasculature allows the shedding of neoplastic cells into thesystemic circulation. Hence, the angiogenic potential of a neoplasmcorrelates with metastatic potential. (Weidner et al. (1991) N. Engl. J.Med. 324(1):1-8; Folkman and Shing (1992) J. Biol. Chem. 267(16):10931-10934).

As a significant proportion of neoplasms are dependent on continuedangiogenesis, inhibition of angiogenesis blocks neoplasm growth whichoften leads to complete necrosis of the neoplasm. (Weidner et al. (1991)N. Engl. J. Med. 324(1):1-8; Folkman and Shing (1992) J. Biol. Chem.267(16):10931-10934). Suppression of anyone of the steps of and/orfactors involved in angiogenesis could inhibit the formation of newvessels, and therefore, affect tumor growth and generation ofmetastases. Indeed, it has been estimated that the elimination of asingle endothelial cell could inhibit the growth of 100 tumor cells(Thorpe et al., 1995). It has also been found that antibodies raisedagainst the angiogenic factor VEGF have been shown to suppress tumorgrowth in vivo (Kim et al., 1993).

As part of treating and managing patients with cancer and many medicalconditions, opioid agonists, such as morphine, are widely used forassociated pain. For example, morphine is used in the terminal phase ofcare of approximately one-half of the patients that die of cancer eachyear in the United States. Opioid agonists, such as morphine comprise agroup of compounds that act on a series of endogenous opioid receptors,such as mu-, kappa-, and delta-receptors in biological systems.Normally, these endogenous receptors bind endogenous opioids. Endogenousopioids are natively produced by mammalian cells.

Endogenous opioids include beta-endorphins, enkephalins, and dynorphins.Beta-endorphins show a preference for mu receptors, enkephalins fordelta receptors and dynorphins for kappa receptors. Opioid agonists areclassified by their preferential effects on the endogenous opioidreceptors. Generally, the mu-receptor is associated with pain relief,and chemical dependence (e.g., drug addiction and alcoholism). Morphine,for example, is a mu-opioid agonist. Opioid receptors are not limited tothe brain and central nervous system (eNS), e.g., to central receptors.Peripheral opioid receptors may be found in other tissues throughout thebody, e.g., gastrointestinal tissue.

Despite wide use in pain management, morphine and other opioidmedications can have severe side effects that may be caused byactivation of the peripheral receptors. The side effects can bedifficult to manage and can result in the patient refusing opioid-basedpain management. Side effects of opioid treatment include nausea,constipation, inhibition of gastrointestinal motility, respiratorysuppression and immunosuppression. Additionally, morphine and otheropioid receptor agonists can stimulate human microvascular endothelialcell proliferation and angiogenesis in vitro and in vivo at typicalmorphine or morphine-equivalent blood concentrations. Thispro-angiogenesis activity of the opioid agonists, while palliative forpain, may hasten tumor progression.

Opioid antagonists are similarly classified by their effects on theopioid receptors, e.g., by their ability to antagonize one receptor moreeffectively than another receptor. For example, the opioid antagonistnaloxone acts as a competitive antagonist at all opioid receptors, butis approximately ten times more effective at mu-receptors than at kappareceptors, and is, therefore, classified as a mu-opioid antagonist.Opioid antagonists may antagonize central receptors, peripheralreceptors or both. Opioid antagonists, and in particular peripheralopioid antagonists, have been used to lessen the side-effects ofexogenously administered opioids, as well as to lessen the unwantedeffects of excessive endogenous opioids.

Opioid antagonists also have been examined for their potential use asanticancer agents for particular types of cancer, as described in U.S.Pat. Nos. 6,384,044 and 6,136,780 and in the scientific literature Guptaet al. Cancer Research, 62: 4491-98 (2002). The anticancer effects ofopioid antagonists have been controversial and not well understood, butit has been held that the opioid antagonist anticancer effects, to theextent they have been shown at all, are unrelated to angiogenesis(Poonawala T, et al., Wound Repair Regen. 2005 March-April; 13(2):165-74; Popov I., Acta Chirlugosl. 2004; 51(2): 117-21; Blebea J, etal., J Vasc Surg. 2002 March; 35(3):532-8; Balasubramanian S, et al., JMol Cell Cardiol. 2001 December; 33(12):2179-87; Zagon I S, et al., IntJ Oncol. 2000 November; 17(5):1053-61; Blebea J et al., J Vasc Surg.2000 August; 32(2):364-73; Pasi A, et al., Gen Pharmacol. (991;22(6):1077-9.) In fact, it has been reported that in xenograft tumormodel in mice, the opioid antagonist naloxone did not exhibit asignificant effect on morphine induced angiogenesis Gupta et al. CancerResearch, 62: 4491-98 (2002). Therefore, it is surprising that it is nowdiscovered that opioid antagonists can inhibit endothelial proliferationand migration associated with angiogenesis.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides methods of attenuating, e.g., inhibiting orreducing, cellular proliferation and migration, particularly endothelialcell proliferation and migration, including that associated withangiogenesis, using opioid antagonists, including, but not limited to,those that are peripherally restricted antagonists.

According to one aspect of the invention, a method of treatment isprovided. The method involves administering to a subject with a disordercharacterized by unwanted migration or proliferation of endothelialcells an effective amount of an opioid antagonist. The treatment mayinhibit one or both of migration and proliferation. The unwantedmigration or proliferation of endothelial cells can be unwantedmigration or proliferation of vascular endothelial cells, including, butnot limited to, unwanted neovascularization or angiogenesis. Examples ofunwanted neovascularization include, but are not limited to,neovascularization associated with cancer and ocular neovascularization.The disorder can be any disorder characterized by unwanted migration orproliferation of endothelial cells. Important such disorders are cancer,sickle cell anemia, vascular wounds, proliferative retinopathies, andunwanted endothelial cell proliferation in the kidneys and the lung.

In important embodiments, the opioid antagonist is a peripheral opioidantagonist. Peripheral opioid antagonists include, but are not limitedto, quaternary or tertiary morphinan derivatives,piperidine-N-alkylcarboxylates, and quaternary benzomorphans. Oneimportant such peripheral opioid antagonist is methylnaltrexone. Anotheropioid antagonist is alvimopan. In important embodiments, the effectiveamount is such that the subject has effective circulating blood plasmalevels of the opioid antagonist continuously for at least 1 week, atleast 2 weeks, at least three weeks and, preferably, at least 4 weeks.

The invention also includes the coadministration of the opioidantagonists with agents that are not opioid antagonists, but which arenonetheless useful in treating disorders characterized by unwantedmigration or proliferation of endothelial cells. Examples of such agentsinclude anticancer agents, antineovascularization agents (for example,anti-VEGF monoclonal antibody), antidiabetes agents, anti-sickle cellagents, wound healing agents, and anthendothelial cell proliferativeagents.

It will be understood that the subjects may be, or may not be, onconcurrent opioid therapy, depending on the particular disorder thesubject has, the severity of the disorder, and the need the subject hasfor pain management. In some embodiments, the subject is takingconcurrent opioid therapy. In some embodiments, the subject is nottaking concurrent opioid therapy. In some embodiments, the subject istaking concurrent chronic opioid therapy. In some embodiments, thesubject is not taking concurrent chronic opioid therapy.

According to another aspect of the invention, a method of inhibitingVEGF activity in endothelial cells is provided. The method involvescontacting the cells with an effective amount of an opioid antagonist.

According to another aspect of the invention, a method of inhibitingexogenous opioid-induced cellular migration or proliferation inendothelial cells is provided. The method involves contacting the cellswith an effective amount of an opioid antagonist.

According to another aspect of the invention, a method of inhibiting RhoA activation in endothelial cells is provided. The method involvescontacting the cells with an effective amount of an opioid antagonist.

According to any of the foregoing embodiments, the opioid antagonistpreferably is a peripheral opioid antagonist, and most preferably ismethylnaltrexone.

The invention provides methods of attenuating migration and/orproliferation of endothelial cells of a tumor or cancer, comprisingcontacting the cells with an antimigratory or antiproliferative amountof an opioid antagonist. In another aspect, the invention providesmethods of attenuating angiogenesis associated with cancer. Thus, theinvention contemplates treating a human cancer patient, for example, bya method of attenuating angiogenesis in a cancerous tissue of a patient,comprising administering to the cancer tissue of the patient aneffective amount of an opioid antagonist.

The invention also provides a method of treating abnormalneovascularization, comprising administering to a patient in need ofsuch treatment, an amount of an opioid antagonist effective to inhibitthe formation of blood vessels.

The invention also includes a method of attenuating tumor progressionand metastasis in animal tissues, comprising contacting tumor cells ortissues with a growth-inhibiting amount of an opioid antagonist, and amethod of attenuating proliferation of hyperproliferative cells in asubject, comprising administering to the subject at least one opioidantagonist, in an amount which is effective to attenuate proliferationof the hyperproliferative cells. In one embodiment, the method involvesadministering a peripheral opioid antagonist, and, in particular, aquaternary derivative of noroxymorphone, to a subject with cancer,whether or not the cancer involves angiogenesis, to treat or inhibit thedevelopment or recurrence of the cancer. Cancers not involvingangiogenesis include those that do not involve the formation of a solidtumor fed by neovasculature. Certain blood cell cancers fall into thiscategory, for example: leukemias (cancer of the leukocytes or whitecells), lymphomas (arising in the lymph nodes or lymphocytes), and somecancers of the bone marrow elements. Thus, in one aspect of theinvention, a method of treatment is provided. The method involvesadministering to a subject with a disorder characterized byhyperproliferation of cells an effective amount of a peripheral opioidantagonist. In one embodiment, the cells are cancer cells. The cancercells may be cancer cells associated with angiogenesis or they may beunassociated with angiogenesis. In one embodiment, the peripheral opioidantagonist is methylnaltrexone.

In further embodiments, the invention provides methods of treatingcancer, wherein a peripheral opioid antagonist and at least one othertherapeutic agent that is not an opioid or opioid antagonist areco-administered to the patient. Suitable therapeutic agents includeanticancer agents (including chemotherapeutic agents and antineoplasticagents), as well as other antiangiogenesis agents. It has beendiscovered that opioid antagonists co-administered with variousanticancer drugs, radiotherapy or other antiangiogenic drugs can giverise to a significantly enhanced antiproliferative effect on cancerouscells, thus providing an increased therapeutic effect, e.g., employingperipheral opioid antagonists to certain tumors can potentiate theirresponse to other therapeutic regimens. Specifically, a significantlyincreased antiproliferative effect, including but not limited to asignificantly increased antiangiogenic effect, is obtained withco-administered combinations as described in more detail below. Not onlycan an existing regimen be enhanced, but new regimens are possible,resulting, for example, in lower concentrations of the anticancercompound, a lower dosing of radiation, or lower concentration of otherantiangiogenic drugs, compared to the treatment regimes in which thedrugs or radiation are used alone. There is the potential, therefore, toprovide therapy wherein adverse side effects associated with theanticancer or other antiangiogenic drugs or radiotherapy areconsiderably reduced than normally observed with the anticancer or otherantiangiogenic drugs or radiotherapy when used alone. Thus, in oneaspect of the invention, a method of treatment is provided. The methodinvolves administering to a subject with a disorder characterized byhyperproliferation of cells an effective amount of an opioid antagonistand an anticancer agent, radiation, or an antiangiogenic agent.

In one embodiment, the cells are cancer cells. In one embodiment, theopioid antagonist is a peripheral opioid antagonist. In one embodiment,the peripheral opioid antagonist is methylnaltrexone. In another aspectof the invention, a method of reducing the risk of recurrence of acancer in a subject after medical intervention is provided. The methodinvolves administering to the subject before, during or after themedical intervention an effective amount of an opioid antagonist and ananticancer agent, radiation, or an antiangiogenic agent. In oneembodiment, the opioid antagonist is a peripheral opioid antagonist. Inone embodiment, the peripheral opioid antagonist is methylnaltrexone.

In another aspect of the invention, the opioid antagonists are usedperi-operatively. By “pen-operatively,” it is meant immediately before(e.g., in preparation for), during, and/or immediately after a surgeryor a surgical or endoscopic procedure, e.g. colonoscopy,gastrolaparoscopy, and especially a surgery or surgical procedureinvolving the removal of a tumor. The opioid antagonists act toattenuate the recurrence of and/or the metastasis of the tumor,especially that arising from angiogenesis associated therewith. It isanticipated that the opioid antagonist will preferably be given in acontinuous dosing regimen, e.g., a regimen that maintains a minimum, andeven more preferably relatively constant, blood level. It is furthercontemplated that the methods of the present invention may haveprophylactic value in certain disorders associated with abnormalangiogenesis. Thus, the invention provides a method of preventing theappearance or re-appearance of a disorder in a mammal, the disorderbeing characterized by unwanted endothelial cell migration orproliferation, including abnormal angiogenesis, comprising administeringto a mammal in need of such treatment, an effective amount of an opioidantagonist, wherein the disorder is a cancer, sickle cell anemia, ocularneovascular diseases, diabetes, ocular retinopathy, or other unwantedendothelial proliferation in kidneys, eye or lung. It will therefore beunderstood that, as used herein, treating a subject with a disordercharacterized by unwanted endothelial cell proliferation or migrationincludes treating a subject with an active disorder to inhibit or curethe disorder and treating a subject to inhibit a disorder fromreoccurring. For example, the subject may have had a solid tumorremoved, and the subject may receive the treatment to inhibit the tumorfrom reoccurring.

In attenuating cell proliferation, the invention provides a method forthe treatment of abnormal cell proliferation of a cell expressingvascular endothelial growth factor (VEGF) in a mammal which comprisesadministering to the mammal a therapeutically effective amount of anopioid antagonist. The invention also includes a method of treatingcancerous tissue in a subject comprising, administering to the subjectan amount of an opioid antagonist sufficient to inhibit VEGF productionin the cancerous tissue, as well as a method of treating angiogenicdisease, the method comprising contacting a tissue or a population ofendothelial cells with a composition comprising an amount of at leastone of an opioid antagonist under conditions effective to inhibitVEGF-induced angiogenesis and to treat angiogenic disease.

In another aspect, the present invention provides a method of inhibitingor reducing angiogenesis, particularly opioid-induced angiogenesis,e.g., of tumor cells, by administrating or providing an opioidantagonist, particularly a peripheral opioid antagonist, to cellsundergoing angiogenesis. In further aspect, the invention providesmethods of treating opioid-induced angiogenesis in patients receivingopioid treatment or in patients where the angiogenesis is induced byendogenous opioids. The former group is typically cancer patients onopioid-based pain management. The methods comprise administering anopioid antagonist to a patient in an antiangiogenic amount, e.g., anamount sufficient to inhibit or reduce the opioid-induced angiogenesis.In those patients receiving opioid treatment, the opioid and theperipheral opioid antagonist may be co-administered. Peripheral opioidantagonists can, thus, be used to inhibit or reduce the angiogeniceffects of opioids on tumor cells, and attenuate the growth of a tumor.

Suitable opioid antagonists generally include heterocyclic aminecompounds that belong to several different classes of compounds. Forexample, one class is suitably tertiary derivatives of morphinan, and inparticular, tertiary derivatives of noroxymorphone. In one embodiment,the tertiary derivative of noroxymorphone is, e.g. naloxone ornaltrexone.

Suitable peripheral opioid antagonists are also generally heterocyclicamine compounds that may belong to several different classes ofcompounds. For example, one class is suitably quaternary derivatives ofmorphinan, and in particular, quaternary derivatives of noroxymorphone.In one embodiment, the quaternary derivative of noroxymorphone is, e.g.,N-methylnaltrexone (or simply methylnaltrexone). Another class isN-substituted piperidines. In one embodiment, the N-piperidine is apiperidine-N-alkylcarbonylate, such as, e.g., alvimopan. Another classof compounds which may be of value in the methods of the presentinvention is quaternary derivatives of benzomorphans.

In some embodiments of the invention, the opioid antagonist may be a muopioid antagonist. In other embodiments, the opioid antagonist may be akappa opioid antagonist. The invention also encompasses administrationof more than one opioid antagonist, including combinations of muantagonists, combinations of kappa antagonists and combinations of muand kappa antagonists, for example, a combination of methylnaltrexoneand alvimopan, or a combination of naltrexone and methylnaltrexone.

In further embodiments, the invention provides methods of treatingopioid-induced angiogenesis in patients receiving an opioid, wherein aperipheral opioid antagonist and at least one other therapeutic agentthat is not an opioid or opioid antagonist are co-administered to thepatient. Suitable therapeutic agents include anticancer agents(including chemotherapeutic agents and antineoplastic agents), as wellas other antiangiogenesis agents.

In yet another aspect, the invention provides a method of reducing therisk of recurrence of a cancer or tumor after medical intervention (suchintervention to include but not be limited to surgery, e.g. pulmonarysurgery, surgical and endoscopic procedures, e.g. colonoscopy,gastrolaparoscopy, chemotherapy, etc.), comprising co-administering to acancer patient an opioid antagonist. Thus, the invention contemplates,for example, a method of minimizing the post-operative recurrence of,e.g., breast cancer in a patient, comprising administering to thepatient an effective amount of an opioid antagonist. Peripheral opioidantagonists in accordance with the present invention, e.g., MNTX, canalso inhibit VEGF, platelet-derived growth factor (PDGF), or sphingosineI-phosphate (SIP)-stimulated or induced cell proliferation in theendothelial cells.

Further provided herein is a method of enhancing the efficacy of acancer chemotherapeutic agent in a subject having a cancer, wherein themethod comprises administering the cancer chemotherapeutic agent and anopioid antagonist to the subject.

In some embodiments, the chemotherapeutic agent is a tyrosine kinaseinhibitor. In some embodiments, the tyrosine kinase inhibitor is an EGFRinhibitor. In some embodiments, the tyrosine kinase inhibitor iserlotinib.

In some embodiments, the chemotherapeutic agent is an anti-microtubuleagent. In some embodiments, the anti-microtubule agent is selected fromthe group consisting of: vinorelbine, paclitaxel, docetaxel, and acombination thereof.

In some embodiments, the chemotherapeutic agent is an antimetabolite. Insome embodiments, the antimetabolite is gemcitabine.

Also provided herein is a method of inhibiting cancer progression in asubject, comprising administering a therapeutically effective amount ofa chemotherapeutic agent with a therapeutically effective amount of anopioid antagonist. In some embodiments, the chemotherapeutic agent is atyrosine kinase inhibitor, an anti-microtubule agent or anantimetabolite.

In some embodiments, the opioid antagonist is a peripheral opioidantagonist. In some embodiments, the peripheral opioid antagonistcomprises a compound of formula (I). In some embodiments, the peripheralopioid antagonist comprises methylnaltrexone.

BRIEF DESCRIPTION OF DRAWINGS

The invention may be better understood and appreciated by reference tothe detailed description of specific embodiments presented herein inconjunction with the accompanying drawings of which:

FIG. 1 is a bar graph of dose-dependent inhibition of humanmicrovascular endothelial cell (HMVEC) migration, depicting the resultsfrom Example 1.

FIG. 2 is a bar graph of dose-dependent inhibition of humanmicrovascular endothelial cell migration, depicting the results fromExample 2.

FIG. 3 is a bar graph of dose-dependent inhibition of HMVEC migrationusing MNTX and MNTX+DAMGO.

FIG. 4 is a bar graph of dose-dependent inhibition of HMVEC migration 15using naloxone and naloxone+DAMGO.

FIG. 5 is a bar graph of dose-dependent effect of M3G and M6G on HMVECmigration.

FIG. 6 is a photomicrograph that shows morphine induced endothelial cellmigration in the presence and absence of MNTX. Panel A=Control, PanelB=20 MS (morphine sulfate), Panel C=MNTX, and Panel D=MS+MNTX. Arrowsare shown in Panel A to highlight several cells that have successfullymigrated across the membrane.

FIG. 7 is a bar graph of percent proliferation (A) and migration (B) ofhuman pulmonary microvascular endothelial cells in the presence of VEGF,25 morphine and DAMGO with or without MNTX.

FIG. 8 is a panel of immunoblots indicating the tyrosine phosphorylation(activation) of (A) of anti-VEGF R.1 (Flt-1) and 2 (Flk-1) usingimmunoprecipitated VEGF R.1 or 2 and anti-phospho-tyrosine in humanpulmonary microvascular endothelial cells in the presence of VEGF,morphine and DAMGO with or without MNTX and a bar graph (B) of percentproliferation and migration of human pulmonary microvascular endothelialcells in the presence of VEGF, morphine and DAMGO with or without VEGFR. inhibitor.

FIG. 9 is a panel of immunoblots indicating RhoA activation usinganti-RhoA in human pulmonary microvascular endothelial cells in thepresence of VEGF, morphine and DAMGO with or without MNTX (A) or VEGF R.Inhibitor (B).

FIG. 10 is a panel of immunoblots (A) of anti-RhoA of human pulmonarymicrovascular endothelial cells in the presence of scramble siRNA(targeting no known human mRNA sequence) or RhoA siRNA and a bar graphof percent proliferation (B) and migration (C) of human pulmonarymicrovascular endothelial cells in the presence of VEGF, morphine andDAMGO with or without scramble siRNA (targeting no known human mRNAsequence) or RhoA siRNA.

FIG. 11 is a schematic diagram summarizing the mechanism of MNTX effectson angiogenesis.

FIG. 12 is a bar graph of percent proliferation above control ofpulmonary microvascular endothelial cells in the presence of SIP, VEGF,PDGF, morphine and DAMGO with or without MNTX.

FIG. 13 is a bar graph of percent migration above control of pulmonarymicrovascular endothelial cells in the presence of SIP, VEGF, PDGF,morphine and DAMGO with or without MNTX.

FIG. 14 is a bar graph of percent proliferation above control ofpulmonary microvascular endothelial cells in the presence of SIP, VEGF,PDGF, morphine and DAMGO with scramble (control) siRNA or with mu opioidreceptor siRNA.

FIG. 15 is a bar graph of percent migration above control of pulmonary25 microvascular endothelial cells in the presence of SIP, VEGF, PDGF,morphine and DAMGO with scramble (control) siRNA or with mu opioidreceptor siRNA.

FIG. 16 is a panel of immunoblots indicating phosphorylation(activation) of the mu opioid receptor using immunoprecipitated muopioid receptor and (A, C) anti-phospho-serine, (B, D)anti-phospho-threonine of human pulmonary microvascular endothelialcells in the presence of morphine, DAMGO, SIP, VEGF, PDGF with MNTX (C,D) or without MNTX (A, B); (E) is an immunoblot of anti-mu opioidreceptor.

FIG. 17 is an anti-RhoA immunoblot of (A, B) activated RhoA and (C)total RhoA of human pulmonary microvascular endothelial cells in thepresence of morphine, DAMGO, SIP, VEGF, PDGF with MNTX (B) and withoutMNTX (A).

FIG. 18 is a panel of immunoblots of top panel: (A, B)anti-phospho-tyrosine, (C) anti-VEGF R and bottom panel: (A, B)anti-phospho-tyrosine, (C) anti-PDGF R, of human pulmonary microvascularendothelial cells in the presence of morphine, DAMGO, VEGF (top panel)or PDGF (bottom panel) with MNTX (B in each panel) or without MNTX (A ineach panel).

FIG. 19 is a panel of immunoblots indicating tyrosine phosphorylation(activation) of the S1P₃ receptor using immunoprecipitated S1P₃ receptorand (A, B) anti-phospho-tyrosine, (C) anti-S1P₃ R, of human pulmonarymicrovascular endothelial cells in the presence of morphine, DAMGO, andS1P with MNTX (B) or without MNTX (A).

FIG. 20 is a bar graph of percent proliferation above control ofpulmonary microvascular endothelial cells in the presence of S1P, VEGF,PDGF, morphine and DAMGO with scramble (control) siRNA or with RhoAsiRNA.

FIG. 21 is a bar graph of percent migration above control of pulmonary20 microvascular endothelial cells in the presence of S1P, VEGF, PDGF,morphine and DAMGO with scramble (control) siRNA or with RhoA siRNA.

FIG. 22 is an schematic diagram summarizing the mechanism of MNTXeffects on RhoA activation and angiogenesis.

FIG. 23 is a graph of percent proliferation above control ofmicrovascular 25 endothelial cells in the presence of VEGF with MNTX,with 5-FU and with a combination of MNTX and 5-FU.

FIG. 24 is a graph of percent migration above control of microvascularendothelial cells in the presence of VEGF with MNTX, with Bevacizumaband with a combination of MNTX and Bevacizumab.

FIG. 25 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on SW480 human colorectal cancer cell line.

FIG. 26 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on HCT116 human colorectal cancer cell line.

FIG. 27 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on MCF-7 human breast cancer cell line.

FIG. 28 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on non-small lung cancer cell (NSLCC) line.

FIG. 29 is a graph of percent inhibition of NSCLC cells in the presenceof (A) erlotinib, (B) paclitaxel, (C) gemcitabine, (D) vironrelbine, and(E) docetaxel with morphine and combinations of morphine and MNTX.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of attenuating abnormal orundesirable migration and/or proliferation of endothelial cells. Assuch, the invention provides methods for attenuating angiogenesis in atissue or an organ of a subject by the use of opioid antagonists, and anovel approach for treating angiogenic related diseases and otherhyperproliferative diseases in mammals.

For example, as described above, solid tumors rely on the generation ofnew blood vessels for nutrients to reach the cells within the tumor. Thegrowth factors required for angiogenesis can be produced by the tumorcells or alternatively, exogenous factors, such as opioids can stimulatenew blood vessel growth. The present invention by the use of opioidantagonists provides a novel therapeutic approach to the treatment ofsuch tumors, wherein the generation of new blood vessels within thetumor, rather than the tumor cells themselves, is the target. Thistreatment is not likely to lead to the development of resistant tumorcells.

Described herein are opioid antagonists inhibit proliferation andmigration induced by opioids, endogenous or exogenous, and growthfactors, such as VEGF, PDGF, S1P etc. Peripheral opioid antagonists, inparticular, showed a substantial efficacy in inhibiting opioid andgrowth factor induced proliferation and migration of endothelial cells.The peripheral opioid antagonist methylnaltrexone (MNTX) inhibited bothopioid and growth factor induced proliferation and migration in aconcentration dependent manner. In addition, naloxone also inhibitedopioid-induced endothelial migration. It should be noted, however, thatthe naloxone inhibition of DAMGO induced migration of endothelial cellsoccurred at a relatively high, micromolar, concentration of naloxone.Furthermore, it has now been discovered that opioid antagonists, and theperipheral opioid antagonist MNTX in particular, inhibit agonist inducedendothelial cell (EC) proliferation and migration via inhibition ofreceptor phosphorylation and/or transactivation and subsequentinhibition of Rho A activation. The agonists can be opioids, exogenousand/or endogenous, angiogenic factors (VEGF), and other proliferationand/or migration stimulating factors (PDGF, S1P, S1P₃ receptor, RhoA,etc). These results suggest that inhibition of angiogenesis by opioidantagonists can be a useful therapeutic intervention for, among otherdisorders, cancer.

The present invention also provides methods of attenuating abnormal orundesirable proliferation of cancer cells per se. This aspect of theinvention is useful in situations involving the presence or absence ofangiogenic activity. The absence of angiogenic activity is evidenced byone or more of the following characteristics: nonsolid tumors or tumorswhere there is repulsion of existing blood vessels and/or absence ofmicro vessels within the tumor, limited growth, for example up to about1 mm in diameter in vivo, at which time further expansion is stopped,harmless to the host until it switches to an angiogenic phenotype, etc.Nonangiogenic tumors can be completely avascular or they can containempty lumen without red blood cells. The gross difference between thenonangiogenic and angiogenic tumors (i.e. white vs. red tumors) is mostlikely due to the reactive hyperemia that accompanies the onset of bloodflow after the angiogenic switch is completed in a previously hypoxictumor. Examples of nonangiogenic tumor lineages include but are notlimited to breast adenocarcinoma, osteosarcoma, glioblastoma, embryonickidney tumors etc. There are many factors that could play a role intumor dormancy and the rate-determining step for tumor expansion ofnonangiogenic tumors could be governed by lack of angiogenesis and/ordifferentiation programs, tumor cell survival, immune response to thehost etc. Although some nonagiogenic tumors never switch to anangiogenic phenotype, many undergo spontaneous transformation into anangiogenic and harmful phenotype. Therefore, treatment of nonangiogenictumors is of therapeutic significance.

Cancers not involving angiogenesis include those that do not involve theformation of a solid tumor fed by neovasculature. Certain blood cellcancers can fall into this category, for example: leukemias, includingacute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),and hairy cell leukemia; lymphomas (arising in the lymph nodes orlymphocytes) including Hodgkin lymphoma, Burkitt's lymphoma, cutaneouslymphoma, cutaneous T-cell lymphoma, follicular lymphoma, lymphoblasticlymphoma, MALT lymphoma, mantle cell lymphoma, Waldenstrom'smacroglobulinemia, primary central nervous system lymphoma; and somecancers of the bone marrow elements including Ewing's sarcoma andosteosarcoma.

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of the structure and function of the invention set forth inthe following description or illustrated in the appended FIGS of thedrawing. The invention is capable of other embodiments and of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of termssuch as “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the item listed thereafter and equivalentsthereof as well as additional items.

Unless otherwise noted, technical terms are used according toconventional usage. As used herein, however, the following definitionsmay be useful in aiding the skilled practitioner in understanding theinvention:

“Subject” refers to humans, dogs, cats, and horses.

“Chronic opioid use” refers to and is characterized by the need forsubstantially higher levels of opioid to produce the therapeutic benefitas a result of prior opioid use, as is well known in the art. Chronicopioid use as used herein includes daily opioid treatment for a week ormore or intermittent opioid use for at least two weeks.

“Alkyl” refers to an aliphatic hydrocarbon group which is saturated andwhich may be straight, branched or cyclic having from 1 to about 10carbon atoms in the chain, and all combinations and subcombinations ofchains therein. Exemplary alkyl groups include methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, see-butyl, tert-butyl, pentyl, hexyl,heptyl, octyl, nonyl and decyl.

“Lower alkyl” refers to an alkyl group having 1 to about 6 carbon atoms.

“Alkenyl” refers to an aliphatic hydrocarbon group containing at leastone carbon-carbon double bond and having from 2 to about 10 carbon atomsin the chain, and all combinations and sub combinations of chainstherein. Exemplary alkenyl groups include vinyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups.

“Alkynyl” refers to an aliphatic hydrocarbon group containing at leastone carbon-carbon triple bond and having from 2 to about 10 carbon atomsin the chain, and combinations and sub combinations of chains therein.Exemplary alkynyl groups include ethynyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl and decenyl groups.

“Alkylene” refers to a bivalent aliphatic hydrocarbon group having from1 to about 6 carbon atoms, and all combinations and subcombinations ofchains therein. The alkylene group may be straight, branched or cyclic.There may be optionally inserted along the alkylene group one or moreoxygen, sulfur or optionally substituted nitrogen atoms, wherein thenitrogen substituent is alkyl as described previously.

“Alkenylene” refers to an alkylene group containing at least onecarbon-carbon double bond. Exemplary alkenylene groups includeethenylene (—CH═CH—) and propenylene (CH═CHCH₂—).

“Cycloalkyl” refers to any stable monocyclic or bicyclic ring havingfrom about 3 to about 10 carbons, and all combinations andsubcombinations of rings therein. The cycloalkyl group may be optionallysubstituted with one or more cycloalkyl-group substituents. Exemplarycycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl groups.

“Cycloalkyl-substituted alkyl” refers to a linear alkyl group,preferably a lower alkyl group, substituted at a terminal carbon with acycloalkyl group, preferably a C3-C8 cycloalkyl group. Exemplarycycloalkyl-substituted alkyl groups include cyclohexylmethyl,cyclohexylethyl, cyclopentylethyl, cyclopentylpropyl, cyclopropylmethyland the like.

“Cycloalkenyl” refers to an olefinically unsaturated cycloaliphaticgroup having from about 4 to about 10 carbons, and all combinations andsubcombinations of rings therein.

“Alkoxy” refers to an alkyl-O-group where alkyl is as previouslydescribed. Exemplary alkoxy groups include, for example, methoxy,ethoxy, propoxy, butoxy and heptoxy.

“Alkoxy-alkyl” refers to an alkyl-O-alkyl group where alkyl is aspreviously described.

“Acyl” means an alkyl-CO group wherein alkyl is as previously described.Exemplary acyl groups include acetyl, propanoyl, 2-methylpropanoyl,butanoyl and palmitoyl.

“Aryl” refers to an aromatic carbocyclic radical containing from about 6to about 10 carbons, and all combinations and subcombinations of ringstherein. The aryl group may be optionally substituted with one or two ormore aryl group substituents. Exemplary aryl groups include phenyl andnaphthyl.

“Aryl-substituted alkyl” refers to a linear alkyl group, preferably alower alkyl group, substituted at a terminal carbon with an optionallysubstituted aryl group, preferably an optionally substituted phenylring. Exemplary aryl-substituted alkyl groups include, for example,phenylmethyl, phenyl ethyl and 3-(4-methylphenyl)propyl.

“Heterocyclic” refers to a monocyclic or multicyclic ring systemcarbocyclic radical containing from about 4 to about 10 members, and allcombinations and subcombinations of rings therein, wherein one or moreof the members of the ring is an element other than carbon, for example,nitrogen, oxygen or sulfur. The heterocyclic group may be aromatic ornonaromatic. Exemplary heterocyclic groups include, for example, pyrroleand piperidine groups.

“Halo” refers to fluoro, chloro, bromo or iodo.

“Peripheral,” in reference to opioid antagonists, designates opioidantagonists that act primarily on physiological systems and componentsexternal to the central nervous system, e.g., they do not readily crossthe blood-brain barrier in an amount effective to inhibit the centraleffects of opioids. In other words, peripheral opioid antagonists do noteffectively inhibit the analgesic effects of opioids when administeredperipherally, e.g., they do not reduce the analgesic effect of theopioids. For example, the peripheral opioid antagonist compoundsemployed in the methods of the present invention exhibit high levels ofactivity with respect to gastrointestinal tissue, while exhibitingreduced or substantially no central nervous system (CNS) activity. Theperipheral opioid antagonist compounds employed in the present methodssuitably exhibit less than about 15% of their pharmacological activityin the CNS, with about 0% (e.g., no CNS activity) being most suitable.The non-central acting characteristic of a peripheral opioid antagonistis often related to charge, polarity and/or size of the molecule. Forexample, peripherally-acting quaternary amine opioid antagonists arepositively charged while the central-acting tertiary amine opioidantagonists are neutral molecules. The peripheral opioid antagonistsuseful in the present invention are typically mu and/or kappa opioidantagonists.

As used herein, “antiangiogenesis” or “antiangiogenic” is meant to referto the capability of a molecule/compound to attenuate, e.g., inhibit,reduce or modulate, proliferation of new blood vessels, in general, andfor example, to reduce or inhibit migration and proliferation of humanmicrovascular endothelial cells in culture in the presence of certaingrowth factors. As described above, the formation of new blood vesselsby endothelial cells involves migration, proliferation anddifferentiation of the cells.

In the following description of the methods of the invention, processsteps are carried out at room temperature and atmospheric pressureunless otherwise specified. It also is specifically understood that anynumerical range recited herein includes all values from the lower valueto the upper value, e.g., all possible combinations of numerical valuesbetween the lowest value and the highest value enumerated are to beconsidered to be expressly stated in this application. For example, if aconcentration range or beneficial effect range is stated as 1% to 50%,it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%,etc., are expressly enumerated in this specification. These are onlyexamples of what is specifically intended.

In one aspect, the present invention relates to methods of attenuatingabnormal or undesirable cellular, particularly endothelial cellmigration and/or proliferation, and angiogenesis in tissue or an organof a subject. The methods comprise providing or administering one ormore opioid antagonists in an effective amount to endothelial cells ofthe tissue or organ of a patient to inhibit endothelial cell migrationand proliferation, and angiogenesis. The angiogenesis may, in part, bethe result of receiving opioid treatment, particularly for painmanagement in cancer patients, or having high levels of endogenousopioids.

It was observed that morphine and the mu agonist enkephalin DAMGO([D-Ala², N-McPhe⁴ Gly⁵-ol]enkephalin), each cause a dose-dependentincrease in migration of endothelial cells similar to that of vascularendothelial growth factor (VEGF) as measured by, e.g., a chemotaxisassay (as detailed in the examples below) or other similar assays usedto identify factors in tumor angiogenesis and the drugs that affect it.At clinically relevant concentrations of morphine, the magnitude of theeffect is approximately 70% of that which is achieved by VEGF. Thismorphine-based endothelial cell migration is attenuated by the mu opioidantagonist methylnaltrexone (MNTX) in a dose-dependent fashion. Forexample, endothelial cell migration induced by morphine, inconcentrations as low as 10⁻⁷M, is significantly blocked by 10⁻⁷M MNTX(FIG. 2). This attenuation strongly suggesting that endothelial cellmigration is mediated by morphine action on the mu opioid receptor(MOR). As described in the examples below, the effect via the MOR ratherthan other opioid receptors is confirmed by experiments that show thehighly selective synthetic enkephalin mu agonist DAMGO also inducesmigration. The migratory effect induced by DAMGO is also blocked by MNTX(FIG. 3).

In one comprehensive review (Neumann et al. Pain 1982; 13:247-52),analgesia in cancer patients was associated with a range of steady stateconcentrations of morphine and plasma ranging from 6 to 364 ng/mL. Itwas observed an effect of morphine that causes endothelial cellmigration at 100 ng/mL well within the clinical dose range. It thereforeis believed by the inventors herein that a dose of MNTX which willmaintain plasma levels of MNTX at minimum levels of plasma MNTX betweenabout 25 and 150 ng/mL would be suitable. Such doses are attainable andare well tolerated (Yuan et al., J Clin Pharmacol 2005; 45:538-46).

Alvimopan, another selective peripheral opioid antagonist given orally,is in late stage development for prophylaxis of postoperative ileus andthe management of opioid induced constipation (Moss et al., Pain reliefwithout side effects: peripheral opioid antagonists. In Schwartz, A J.,editor. 33rd ASA Refresher Course in Anesthesiology. Philadelphia:Lippincott Williams & Wilkins (in press).) There is some transpassage ofalvimopan across the membrane (J. Foss, et al., Clin. Pharm. & Ther.2005, PII-90, p. 74) and it may, therefore, possess the ability toreverse some of the systemic effects of opioids without affectinganalgesia even when given orally.

Without being bound by any particular theory, it may be that themechanism of mu opioid effect on endothelial cell migration occurs atthe membrane level as MNTX, unlike naloxone, is a charged molecule atphysiological pH. Morphine acts via G-protein coupled receptors, whileVEGF acts by receptor tyrosine kinases. While the actions of mu agonistsand VEGF may be independent, there is growing evidence of receptortransactivation as a mechanism. A prior investigation demonstrated thatpertussis toxin dependent GPCRs transactivate VEGF receptor-2/F1 K1(Zeng, H. et al., J. Biol. Chem. 2003; 278:20738-45). By this mannermorphine could transactivate F11 c-1 and promote an environment whereendothelial cell proliferation and tumor growth could occur. A recentstudy of MOR knockout mice infected with T241 fibrosarcoma cellsdemonstrated significant differences in the incidences of tumor growthand a 10-fold increase in F11 c-1 expression in morphine treated miceversus controls, versus no increase in morphine treated KO mice (K.Gupta, personal communication). This provides further evidence thatmorphine stimulates endothelial cell proliferation and promotes tumorgrowth probably by trans activating FLK1 phosphorylation. As such, thepresent invention provides potential clinical strategies using MNTX aswell as other peripheral opioid antagonist in conjunction with currenttherapies targeting VEGF. Although a direct effect by receptortransactivation is possible, a potential additional factor involved inthe proliferation of tumors may well be the role of chemokines asintegrators of pain and inflammation. A recent review on this subject(White et al., Nature Rev. Drug Discovery 2005; 4:834-44) also suggestsa possible role for leukocytes in activating opioid receptors.

Furthermore, it was observed that morphine, DAMGO and VEGF stimulateRhoA activation which is inhibited by opioid antagonists, such as MNTX.RhoA is an important signaling molecule involved in angiogenesis(Aepfelbacher et al., 1997; Cascone et al., 2003; Hoang et al., 2004;Liu and Sanger, 2004.) VEGF receptor transactivation is important foropiate-induced RhoA activation. Silencing RhoA expression blocked opioidand VEGF induced EC proliferation and migration, demonstrating a rolefor RhoA activation in agonist-induced EC angiogenic activity. The MNTXmediated attenuation of RhoA activation may be important for theinhibitory role of MNTX on opioid and VEGF induced angiogenesis.

Because morphine and other opioids at clinical doses enhance endothelialcell migration, the present invention may be of therapeutic value inopioid antagonist treatment for patients on significant and sustaineddoses of opioids that have tumors relying on the angiogenic process.Further, while the inventor's clinical observations have focused onmorphine, which is exogenously administered, endogenous opioids, whichare released by stress or pain, may also play a role in endothelial cellmigration. Based on endothelial cell migration experiments detailedbelow in the examples, MNTX and opioid antagonists generally are oftherapeutic value as an antiangiogenic therapy even absent exogenousopioid administration (as detailed herein). It is envisioned that themethods of the present invention will inhibit or reduce the growth ofblood vessels within and to a tumor. Inhibiting the growth of bloodvessels within tumors prevents nutrients and oxygen from being suppliedto the tumor to support growth beyond a certain size. Minimizing thenumber of blood vessels or other tumors also lessens the probabilitythat the tumor will metastasize.

Multidrug resistance, an important mechanism by which many cancersdevelop resistance to chemotherapeutic drugs, is a major contributingfactor to the failure of cancer drug therapy. It affects patients with avariety of cancers including lung, breast, ovarian and lowergastrointestinal tract cancers. Resistance to therapy is regulated by atleast two classes of molecular pumps in tumor-cell plasma membranes thatactively expel chemotherapeutic drugs from inside the cell. This allowstumor cells to avoid the toxic effects of the drugs and promotesmultidrug resistance. The two pumps commonly found in cancer areP-glycoprotein and the multidrug resistance-associated proteins (MRP)that belong to the ATP-binding cassette (ABC) transporters. A major MRPexpressed in NSCLC is ABCC3. Morphine may promote drug resistance to avariety of chemotherapeutic agents with different mechanisms of actionincluding tyrosine kinase inhibitor, microtubule modifying agents andDNA synthesis inhibitor. Among other things, the present invention maybe useful to reverse the multidrug resistance effects of morphine.

The present invention may be of therapeutic value in opioid antagonisttreatment for patients who have tumors relying on the angiogenicprocess. Tumors that rely on angiogenic processes are solid tumors,leukemias and myelomas. Solid tumors include, but are not limited toadrenal cortical carcinoma, tumors of the bladder: squamous cellcarcinoma, urothelial carcinomas; tumors of the bone: adamantinoma,aneurysmal bone cysts, chondroblastoma, chondroma, chondromyxoidfibroma, chondrosarcoma, fibrous dysplasia of the bone, giant celltumour, osteochondroma, osteosarcoma; breast tumors: secretory ductalcarcinoma, chordoma; colon tumors: colorectal adenocarcinoma; eyetumors: posterior uveal melanoma, fibrogenesis imperfecta ossium, headand neck squamous cell carcinoma; kidney tumors: chromophobe renal cellcarcinoma, clear cell renal cell carcinoma, nephroblastoma (Wilmstumor), kidney: papillary renal cell carcinoma, primary renal ASPS CR1-TFE3 tumor, renal cell carcinoma; liver tumors: hepatoblastoma,hepatocellular carcinoma; lung tumors: non-small cell carcinoma, smallcell cancer; malignant melanoma of soft parts; nervous system tumors:medulloblastoma, meningioma, neuroblastoma, astrocytic tumors,ependymomas, peripheral nerve sheath tumors, phaeochromocytoma; ovariantumors: epithelial tumors, germ cell tumors, sex cord-stromal tumors,pericytoma; pituitary adenomas; rhabdoid tumor; skin tumors: cutaneousbenign fibrous histiocytomas; smooth muscle tumors: intravenousleiomyomatosis; soft tissue tumors: liposarcoma, myxoid liposarcoma, lowgrade fibromyxoid sarcoma, leiomyosarcoma, alveolar soft part sarcoma,angiomatoid fibrous histiocytoma (AFH), clear cell sarcoma, desmoplasticsmall round cell tumor, elastofibroma, Ewing's tumors, extraskeletalmyxoid chondrosarcoma, inflammatory myofibroblastic tumor, lipoblastoma,lipoma/benign lipomatous tumors, liposarcoma/malignant lipomatoustumors, malignant myoepithelioma, rhabdomyosarcoma, synovial sarcoma,squamous cell cancer; tumors of the testis: germ cell tumors,spermatocytic seminoma; thyroid tumors: anaplastic (undifferentiated)carcinoma, oncocytic tumors, papillary carcinoma; uterus tumors:carcinoma of the cervix, endometrial carcinoma, leiomyoma etc.

In one embodiment of the invention the tumors are prostate cancer,gastrointestinal tumors such as colon or pancreatic cancer and thecompounds of the invention are co-administered with other anticanceragents as described herein.

The opioid antagonists in accordance with the present invention includeboth centrally and peripherally acting opioid antagonists. It iscontemplated that those antagonists of particular value are suitably theperipheral opioid antagonists. Especially suitable may be a mu opioidantagonist, especially a mu peripheral opioid antagonist. Opioidantagonists form a class of compounds that can vary in structure whilemaintaining the peripheral restrictive property. These compounds includetertiary and quaternary morphinans, in particular noroxymorphonederivatives, N-substituted piperidines, and in particular,piperidine-N-alkylcarboxylates, and tertiary and quaternarybenzomorphans. Peripherally restricted antagonists, while varied instructure, are typically charged, polar and/or of high molecular weight,each of which impedes their crossing the blood-brain barrier.

Examples of opioid antagonists, which cross the blood-brain barrier andare centrally (and peripherally) active, include, e.g., naloxone,naltrexone (each of which is commercially available from BaxterPharmaceutical Products, Inc.) and nalmefene (available, e.g., fromDuPont Pharma). These may be of value in attenuating angiogenesis in thecentral nervous system or in patients not being treated for painmanagement or other opioid treatment.

A peripheral opioid antagonist useful for the present invention may be acompound which is a quaternary morphinan derivative, and in particular,a quaternary noroxymorphone of formula (I):

wherein R is alkyl, alkenyl, alkynyl, aryl, cycloalkyl-substituted alkylor aryl-substituted alkyl, and X′ is the anion, especially a chloride,bromide, iodide or methylsulfate anion. The noroxymorphone derivativesof formula (I) can be prepared, for example, according to the procedurein U.S. Pat. No. 4,176,186, which is incorporated herein by reference;see also, U.S. Pat. Nos. 4,719,215; 4,861,781; 5,102,887; 5,972,954 and6,274,591, U.S. Patent Application Nos. 2002/0028825 and 2003/0022909;and PCT publication Nos. WO 99/22737 and WO 98/25613, all of which arehereby incorporated by reference.

A compound of formula (I) of particular value is N-methylnaltrexone (orsimply methylnaltrexone), wherein R is cyclopropylmethyl as representedin formula (II):

wherein X— is as described above. Methylnaltrexone is a quaternaryderivative of the opioid antagonist naltrexone. Methylnaltrexone existsas a salt, and “methylnaltrexone” or “MNTX”, as used herein, thereforeembraces salts. “Methylnaltrexone” or “MNTX” specifically includes, butis not limited to, bromide salts, chloride salts, iodide salts,carbonate salts, and sulfate salts of methylnaltrexone. Names used forthe bromide salt of MNTX in the literature include: methylnaltrexonebromide; N-methylnaltrexone bromide; naltrexone methobromide; naltrexonemethyl bromide; SC-37359, MRZ-2663-BR, andN-cyclopropylmethylnoroxy-morphine-methobromide. Methylnaltrexone iscommercially available from, e.g., Mallinclaodt Pharmaceuticals, St.Louis, Mo. Methylnaltrexone is provided as a white crystalline powder,freely soluble in water, typically as the bromide salt. The compound asprovided is 99.4% pure by reverse phase HPLC, and contains less than0.011% unquaternized naltrexone by the same method. Methylnaltrexone canbe prepared as a sterile solution at a concentration of, e.g., about 5mg/mL.

Other suitable peripheral opioid antagonists may include N-substitutedpiperidines, and in particular, piperidine-N-alkylcarboxylates asrepresented by formula (III):

whereinR¹ is hydrogen or alkyl;R² is hydrogen, alkyl, or alkenyl;R³ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl;R⁴ is hydrogen, alkyl, or alkenyl;A is OR⁵ or NR⁶R⁷; whereinR⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl;R⁶ is hydrogen or alkyl;R⁷ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl, or alkylene-substituted B or together with thenitrogen atom to which they are attached, R⁶ and R⁷ form a heterocyclicring selected from pyrrole and piperidine; B is

wherein R⁸ is hydrogen or alkyl;R⁹ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl or together with the nitrogen atom to which theyare attached, R⁸ and R⁹ form a heterocyclic ring selected from pyrroleand piperidine;W is OR¹⁰, NR¹¹R¹², or OE; whereinR¹⁰ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkenyl, oraryl-substituted alkyl;R¹¹ is hydrogen or alkyl;R¹² is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl,aryl-substituted alkyl or alkylene-substituted C(═O)Y or, together withthe nitrogen atom to which they are attached, R¹¹ and R¹² form aheterocyclic ring selected from pyrrole and piperidine;E is

alkylene-substituted (C═O)D, or —R¹³0C(═O)R¹⁴; whereinR¹³ is alkyl-substituted alkylene;R¹⁴ is alkyl;D is OR¹⁶ or NR¹⁶R¹⁷; whereinR¹⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl substituted alkyl, oraryl-substituted alkyl;R¹⁶ is hydrogen, alkyl, alkenyl, aryl, aryl-substituted alkyl,cycloalkyl, cycloalkenyl, cycloalkyl substituted alkyl orcycloalkenyl-substituted alkyl;R¹⁷ is hydrogen or alkyl or, together with the nitrogen atom to whichthey are attached, R¹⁶ and R¹⁷ form a heterocyclic ring selected fromthe group consisting of pyrrole or piperidine;Y is OR¹⁸ or NR¹⁹R²⁰; whereinR¹⁸ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl;R¹⁹ is hydrogen or alkyl;R²⁰ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl or, together with the nitrogen atom to which theyare attached, R¹⁹ and R²⁰ form a heterocyclic ring selected from pyrroleand piperidine;R²¹ is hydrogen or alkyl;and n is 0 to 4.

Particular piperidine-N-alkylcarbonylates which may be of value areN-alkylamino-3,4,4 substituted piperidines, such as alvimopanrepresented below as formula (IV):

Suitable N-substituted piperidines may be prepared as disclosed in U.S.Pat. Nos. 5,270,328; 6,451,806; 6,469,030, all of which are herebyincorporated by reference. Alvimopan is available from Adolor Corp.,Exton, Pa. Such compounds have moderately high molecular weights, azwitterion form and a polarity which prevent penetration of theblood-brain barrier.

Still other suitable peripheral opioid antagonist compounds may includequaternary benzomorphan compounds. The quaternary benzomorphan compoundsemployed in the methods of the present invention exhibit high levels ofmorphine antagonism, while exhibiting reduced, and preferablysubstantially no, agonist activity.

The quaternary benzomorphan compounds which may be employed in themethods of the present invention have the following formula (V):

wherein;R¹ is hydrogen, acyl or acetoxy; andR² is alkyl or alkenyl;R is alkyl, alkenyl or alkynyl andX⁻ is an anion, especially a chloride, bromide, iodide or methylsulfateanion.

Specific quaternary derivatives of benzomorphan compounds that may beemployed in the methods of the present invention include the followingcompounds of formula (V): 2′-hydroxy5,9-dimethyl-2,2-diallyl-6,7-benzomorphanium-bromide;2′-hydroxy-5,9-dimethyl-2-n-propyl-2 ally 1-6,7-benzomorphanium-bromide;2′-hydroxy-5,9-dimethyl-2-n-propyl-2-propargyl-6, 7benzomorphanium-bromide; and2′-acetoxy-5,9-dimethyl-2-n-propyl-2-ally)-6,7 benzomorphanium-bromide.

Other quaternary benzomorphan compounds that may be employed in themethods of the present invention are described, for example, in U.S.Pat. No. 3,723,440, the entire disclosure of which is incorporatedherein by reference. The compounds employed in the methods of thepresent invention may exist in prodrug form. As used herein, “prodrug”is intended to include any covalently bonded carriers which release theactive parent drug according to formulas (I) to (V) or other formulas orcompounds employed in the methods of the present invention in vivo whensuch prodrug is administered to a mammalian subject. Since prodrugs areknown to enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compoundsemployed in the present methods may, if desired, be delivered in prodrugform. Thus, the present invention contemplates methods of deliveringprodrugs. Prodrugs of the compounds employed in the present inventionmay be prepared by modifying functional groups present in the compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, amino, or carboxy group is bonded to any group that,when the prodrug is administered to a mammalian subject, cleaves to forma free hydroxyl, free amino, or carboxylic acid, respectively.

Examples include, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups; and alkyl,carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl,iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl,benzyl, and phenethyl esters, and the like.

As noted, the compounds employed in the methods of the present inventionmay be prepared in a number of ways well known to those skilled in theart. All preparations disclosed in association with the presentinvention are contemplated to be practiced on any scale, includingmilligram, gram, multigram, kilogram, multikilogram or commercialpharmaceutical scale.

Compounds employed in the present methods may contain one or moreasymmetrically substituted carbon atom, and may be isolated in opticallyactive or racemic form. Thus, all chiral, diastereomeric, racemic form,epimeric form and all geometric isomeric form of a structure areintended, unless the specific stereochemistry or isomeric form isspecifically indicated. It is well known in the art how to prepare andisolate such optically active form. For example, mixtures of stereoisomers may be separated by standard techniques including, but notlimited to, resolution of racemic form, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

In some embodiments of the invention, the opioid antagonist may be a muopioid antagonist. In other embodiments, the opioid antagonist may be akappa opioid antagonist. The invention also encompasses administrationof more than one opioid antagonist, including combinations of muantagonists, combinations of kappa antagonists and combinations of muand kappa antagonists, for example, a combination of methylnaltrexoneand alvimopan.

The methods of the present invention encompass providing a therapeuticor prophylactic role in other endothelial-based diseases, e.g., in avariety of angiogenesis and/or proliferation-related neoplastic andnon-neoplastic diseases, e.g., sickle cell disease, neovascular diseaseof the eye (such as diabetic retinopathy, neovascular glaucoma,retinopathy of prematurity, age-related macular degeneration),endothelial proliferation in the kidneys or lung and psoriasis.Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, chronic inflammation, lunginflammation, nephrotic syndrome, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), and pleuraleffusion. For example, it has been shown that morphine inducedproliferative retinopathy in sickle cell disease (Gupta et al., personalcommunication). It is anticipated that treatment with an opioidantagonist may significantly inhibit the retinopathy, particularly withopioid-induced retinopathy in sickle cell patients that are in activeopioid therapy, and receive opioids for long periods of time, includingchronic therapy for weeks, months or even years.

The methods of the present invention are also envisioned to be of valuein reducing the risk of recurrence of a malignancy or neoplasm aftertreatment with other therapeutic modalities, e.g., after surgicalintervention. For example, the present invention provides a method forreducing the risk of recurrence of postoperative cancer. The cancers mayinclude, for example, breast cancer or prostate cancer, and reduced riskmay be achieved by providing to the patient suffering from such canceran effective amount of an opioid antagonist, particularly a peripheralopioid antagonist. For example, as described above, patients undergoingbreast cancer surgery had a significant difference (fourfold) in theincidence of recurrence at 2-4 years depending on whether the patientsreceived regional or general anesthesia (with morphine during theirinitial surgery. Co-administration of the opioid antagonists, especiallyperipheral antagonist, in accordance with the present invention withsurgical treatment may be of value to reduce the incidence of recurrenceof the cancer.

It is also contemplated that the invention provides a method ofinhibiting the activity of VEGF by providing to the affected cells orsubject an effective amount of an opioid antagonist under conditionssufficient to inhibit VEGF-induced angiogenesis. In other words, thecompounds of the present invention have VEGF-inhibitory or antagonistactivity.

As also shown in the examples below, it has further been found that aperipheral opioid antagonist, MNTX, attenuates not only VEGF-inducedendothelial cell migration, but also induction of endothelial migrationand/or proliferation by other pro-migration/pro-proliferative factorssuch as platelet derived growth factor (PDGF), or sphingosine1-phosphate (S1P). Such attenuation ranges from about 10% to 60%, andprovides further evidence that the methods of the present invention havevalue in inhibiting pro-migration, pro-angiogenic factors.

The methods of the invention also encompass treating patients, e.g.,cancer patients, who are undergoing treatment with opioid agonists.Opioid agonists include, but are not limited to, morphine, methadone,codeine, meperidine, fentidine, fentanil, sufentanil, alfentanil and thelike. As described above, opioid agonists are classified by theirability to agonize one type of receptor an order of magnitude moreeffectively than another. For example, the relative affinity of morphinefor the mu receptor is 200 times greater than for the kappa receptor,and is therefore classified as a mu opioid agonist. Some opioid agonistsmay act as agonists towards one receptor and antagonists toward anotherreceptor and are classified as agonist/antagonists, (also known as mixedor partial agonists). “Agonist/antagonist,” “partial agonist,” and“mixed agonist” are used interchangeably herein. These opioids include,but are not limited to, pentazocine, butorphanol, nalorphine, nalbufine,buprenorphine, bremazocine, and bezocine. Many of the agonist/antagonistgroup of opioids are agonists at the kappa receptors and antagonists atthe mu receptors. Further, it is envisioned the active metabolites ofopioid agonists may also be active as angiogenesis inducers. Forexample, the metabolites of morphine, morphine 3-glucuronide (M3G) andmorphine 6-glucuronide (M6G) may be active proangiogenic factors.

Generally, the peripheral opioid antagonists in accordance with thepresent invention may be administered in an effective amount such thatthe patient's plasma level of the peripheral opioid antagonist is in therange from 10⁻⁶ M to 10⁻⁹M. Patient drug plasma levels may be measuredusing routine HPLC methods known to those of skill in the art.

As described in the examples below, the enkephalin analog DAMGO inducesendothelial migration. Thus, the methods of the present invention may beof value to patient suffering from angiogenic-related orhyperproliferative diseases, e.g., cancer, quite apart from treatmentwith opioid agonists.

The particular mode of administration of the opioid antagonist selectedwill depend, of course, upon the particular combination of drugsselected, the severity of the tumor progression being treated, in thecancer patient, the general health condition of the patient, and thedosage required for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, e.g., any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical (as by powder, ointment, drops, transdermal patch oriontophoretic devise), trans dermal, sublingual, intramuscular,infusion, intravenous, pulmonary, intramuscular, intracavity, as anaerosol, aural (e.g., via eardrops), intranasal, inhalation, intraocularor subcutaneous. Direct injection could also be used for local delivery.Oral or subcutaneous administration may be suitable for prophylactic orlong term treatment because of the convenience of the patient as well asthe dosing schedule. For ocular diseases, ophthalmic formulations may beinjected or instilled directly.

Additionally, the opioid antagonists may be administered as anenterically coated tablet or capsule. In some embodiments, the opioidantagonist is administered by a slow infusion method or by atime-release or controlled-release method or as a lyophilized powder.

When administered, the compounds of the invention are given inpharmaceutically acceptable amounts and in pharmaceutically acceptablecompositions or preparations. Such preparations may routinely containsalts, buffering agents, preservatives, and optionally other therapeuticingredients. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically acceptable salts thereof and are notexcluded from the scope of the invention.

Such pharmacologically and pharmaceutically acceptable salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic,succinic, naphthalene-2-sulfonic, pamoic,3-hydroxy-2-naphthalenecarboxylic, and benzene sulfonic. Suitablebuffering agents include, but are not limited to, acetic acid and saltsthereof (1-2% WN); citric acid and salts thereof (1-3% WN); boric acidand salts thereof (0.5-2.5% WN); and phosphoric acid and salts thereof(0.8-2% WN).

Suitable preservatives include, but are not limited to, benzalkoniumchloride (0.003-0.03% WN); chlorobutanol (0.3-0.9% WIN); parabens(0.01-0.25% WN) and thimerosal (0.004-0.02% WN).

For ease of administration, a pharmaceutical composition of theperipheral opioid antagonist may also contain one or morepharmaceutically acceptable excipients, such as lubricants, diluents,binders, carriers, and disintegrants. Other auxiliary agents mayinclude, e.g., stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, coloring, flavoring and/or aromatic activecompounds.

A pharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. For example, suitablepharmaceutically acceptable carriers, diluents, solvents or vehiclesinclude, but are not limited to, water, salt (buffer) solutions,alcohols, gum arabic, mineral and vegetable oils, benzyl alcohols,polyethylene glycols, gelatin, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin,vegetable oils, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. Proper fluidity may be maintained, for example, by theuse of coating materials such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. Prevention of the action of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like.

If a pharmaceutically acceptable solid carrier is used, the dosage formof the analogs may be tablets, capsules, powders, suppositories, orlozenges. If a liquid carrier is used, soft gelatin capsules,transdermal patches, aerosol sprays, topical cream, syrups or liquidsuspensions, emulsions or solutions may be the dosage form.

For parental application, particularly suitable are injectable, sterilesolutions, preferably nonaqueous or aqueous solutions, as well asdispersions, suspensions, emulsions, or implants, includingsuppositories. Ampoules are often convenient unit dosages. Injectabledepot form may also be suitable and may be made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.

Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules such as soft gelatincapsules. A syrup, elixir, or the like can be used wherein a sweetenedvehicle is employed.

As noted, other delivery system may include time-release,delayed-release or sustained-release delivery system. Such system canavoid repeated administrations of the compounds of the invention,increasing convenience to the patient and the physician and maintainsustained plasma levels of compounds. Many types of controlled-releasedelivery system are available and known to those of ordinary skill inthe art. Sustained- or controlled-release compositions can beformulated, e.g., as liposomes or those wherein the active compound isprotected with differentially degradable coatings, such as bymicroencapsulation, multiple coatings, etc.

For example, compounds of this invention may be combined withpharmaceutically acceptable sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions. Asustained-release matrix, as used herein, is a matrix made of materials,usually polymers, which are degradable by enzymatic or acid-basehydrolysis or by dissolution. Once inserted into the body, the matrix isacted upon by enzymes and body fluids. A sustained-release matrix may bedesirably chosen from biocompatible materials such as liposomes,polymer-based system such as polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(copolymers of lactic acid and glycolic acid), polyanhydrides,poly(ortho)esters, polysaccharides, polyamino acids, hyaluronic acid,collagen, chondroitin sulfate, polynucleotides, polyvinyl propylene,polyvinyl pyrrolidone, and silicone; nonpolymer system such ascarboxylic acids, fatty acids, phospholipids, amino acids, lipids suchas sterols, hydrogel release system; silastic system; peptide-basedsystem; implants and the like. Specific examples include, but are notlimited to: (a) erosional system in which the polysaccharide iscontained in a form within a matrix, found in U.S. Pat. Nos. 4,452,775,4,675,189, and 5,736,152 (herein incorporated by reference in theirentireties), and (b) diffusional system in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686 (herein incorporated byreference in their entireties). In addition, pump-based hard-wireddelivery system can be used, some of which are adapted for implantation.Suitable enteric coatings are described in PCT publication No. WO98/25613 and U.S. Pat. No. 6,274,591, both incorporated herein byreference.

Use of a long-term sustained-release implant may be particularlysuitable for treatment of chronic conditions. “Long-term” release, asused herein, means that the implant is constructed and arranged todeliver therapeutic levels of the active ingredient for at least 7 days,and suitably 30 to 60 days. Long-term sustained-release implants arewell-known to those of ordinary skill in the art and include some of therelease system described above.

For topical application, there are employed as nonsprayable form,viscous to semi-solid or solid form comprising a carrier compatible withtopical application and having a dynamic viscosity preferably greaterthan water. Suitable formulations include, but are not limited to,solutions, suspensions, emulsions, cream, ointments, powders, liniments,salves, aerosols, etc., which are, if desired, sterilized or mixed withauxiliary agents, e.g., preservatives, etc.

Transdermal or iontophoretic delivery of pharmaceutical compositions ofthe peripheral opioid antagonists is also possible.

Respecting MNTX specifically, aqueous formulations may include chelatingagent, a buffering agent, an anti-oxidant and, optionally, anisotonicity agent, preferably pH adjusted to between 3.0 and 3.5.Preferred such formulations that are stable to autoclaving and long termstorage are described in application Ser. No. 10/821,811, now publishedas US 2004/0266806, entitled “Pharmaceutical Formulation,” thedisclosure of which is incorporated herein by reference.

In one embodiment, compounds of the invention are administered in adosing regimen which provides a continuous dosing regimen of thecompound to a subject, e.g., a regimen that maintains minimum plasmalevels of the opioid antagonist, and preferably eliminates the spikesand troughs of a drug level with conventional regimens. Suitably, acontinuous dose may be achieved by administering the compound to asubject on a daily basis using any of the delivery methods disclosedherein. In one embodiment, the continuous dose may be achieved usingcontinuous infusion to the subject, or via a mechanism that facilitatesthe release of the compound over time, for example, a transdermal patch,or a sustained release formulation. Suitably, compounds of the inventionare continuously released to the subject in amounts sufficient tomaintain a concentration of the compound in the plasma of the subjecteffective to inhibit or reduce opioid induced angiogenesis; or in cancerpatients, to attenuate growth of a tumor. Compounds in accordance withthe present invention, whether provided alone or in combination withother therapeutic agents, are provided in an antiangiogenic effectiveamount. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the level of ordinary skill in the art to start doses of thecompound at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. As noted, those of ordinary skill in the art will readily optimizeeffective doses and co-administration regimens (as described herein) asdetermined by good medical practice and the clinical condition of theindividual patient.

Generally, oral doses of the opioid antagonists, particularly peripheralantagonists, will range from about 0.01 to about 80 mg/kg body weightper day. It is expected that oral doses in the range from 1 to 20 mg/kgbody weight will yield the desired results. Generally, parenteraladministration, including intravenous and subcutaneous administration,will range from about 0.001 to 5 mg/kg body weight. It is expected thatdoses ranging from 0.05 to 0.5 mg/kg body weight will yield the desiredresults. Dosage may be adjusted appropriately to achieve desired druglevels, local or systemic, depending on the mode of administration. Forexample, it is expected that the dosage for oral administration of theopioid antagonists in an enteric ally coated formulation would be from10 to 30% of the non-coated oral dose. In the event that the response ina patient is insufficient of such doses, even higher doses (oreffectively higher 30 dosage by a different, more localized deliveryroute) may be employed to the extent that the patient tolerance permits.Multiple doses per day are contemplated to achieve appropriate systemiclevels of compounds. Appropriate system levels can be determined by, forexample, measurement of the patient's plasma level of the drug usingroutine HPLC methods known to these of skill in the art.

In some embodiments of the invention, the opioid antagonists areco-administered with the opioid. The term “co-administration” is meantto refer to a combination therapy by any administration route in whichtwo or more agents are administered to a patient or subject.Co-administration of agents may also be referred to as combinationtherapy or combination treatment. The agents may be in the same dosageformulations or separate formulations. For combination treatment withmore than one active agent, where the active agents are in separatedosage formulations, the active agents can be administered concurrently,or they each can be administered at separately staggered times. Theagents may be administered simultaneously or sequentially (e.g., oneagent may directly follow administration of the other or the agents maybe give episodically, e.g., one can be given at one time followed by theother at a later time, e.g., within a week), as long as they are givenin a manner sufficient to allow both agents to achieve effectiveconcentrations in the body. The agents may also be administered bydifferent routes, e.g., one agent may be administered intravenouslywhile a second agent is administered intramuscularly, intravenously ororally. In other words, the co-administration of the opioid antagonistcompound in accordance with the present invention with an opioid issuitably considered a combined pharmaceutical preparation which containsan opioid antagonist and a opioid agent, the preparation being adaptedfor the administration of the peripheral opioid antagonist on a daily orintermittent basis, and the administration of opioid agent on a daily orintermittent basis. Thus, the opioid antagonists may be administeredprior to, concomitant with, or after administration of the opioids.Co-administrable agents also may be formulated as an admixture, as, forexample, in a single formulation or single tablet. These formulationsmay be parenteral or oral, such as the formulations described, e.g., inU.S. Pat. Nos. 6,277,384; 6,261,599; 5,958,452 and PCT publication No.WO 98/25613, each hereby incorporated by reference.

It is further contemplated that the present method can be used alone orin conjunction with other treatments to control the growth or migrationof endothelial cells in connection with the various conditions describedabove. The peripheral opioid antagonist may be co-administered withanother therapeutic agent that is not an opioid or opioid antagonist.Suitable such therapeutic agents include anticancer agents, e.g.,chemotherapeutic agents, radiotherapy, or other antiangiogenic agentssuch as suramin, or anti-VEGF mab, an endostatin or radiotherapy. It isenvisioned that the opioid antagonists in accordance with the presentinvention are of particular value when co-administered with those agentsthat inhibit VEGF activity, e.g., anti-VEGF mab. The anti-VEGFantibodies are useful in the treatment of various neoplastic andnon-neoplastic diseases and disorders, including endometrialhyperplasia, endometriosis, abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors andMeigs' syndrome. One example of an anti-VEGF mab is bevacizumab(Avastin, Genentech) described in U.S. Pat. No. 6,884,879 and WO94/10202hereby incorporated in their entirety. In a certain embodiments of theinvention, MNTX is co-administered with Avastin.

In other words, the compounds of the present invention may also beuseful for the treatment of cancer in patients, as described above,either when used alone or in combination with one or more otheranticancer agents, e.g., radiotherapy and/or other chemotherapeutic,including antiangiogenic, treatments conventionally administered topatients for treating cancer. The main categories and examples of suchdrugs are listed herein and include, but are not limited tometalloprotease inhibitors, inhibitors of endothelial cellproliferation/migration, antagonists of angiogenic growth factors,inhibitors of Integrin/Survival signaling, and chelators of copper.

In certain embodiments the compounds of the invention can be combinedwith known combinations of anticancer agents. The compounds of theinvention can be combined with an antiangiogenic agent and achemotherapeutic agent and administered to a cancer patient. Forexample, MNTX can be administered to cancer patients in combination withAvastin and 5-fluorouracil. It is anticipated that the opioidantagonists co-administered with various anticancer drugs, radiotherapyor other anti angiogenic drugs can give rise to a significantly enhancedantiproliferative effect on cancerous cells, and thus providing anincreased therapeutic effect, e.g., employing peripheral opioidantagonists to certain tumors can potentiate their response to othertherapeutic regimens. Specifically, a significantly increased antiangiogenic or antihyperproliferative effect is obtained with the abovedisclosed co-administered combinations, even if utilizing lowerconcentrations of the anticancer, a lower dosing of radiation, or otherantiangiogenic drugs compared to the treatment regimes in which thedrugs or radiation are used alone. Therefore there is the potential toprovide therapy wherein adverse side effects associated with theanticancer or other antiangiogenic drugs or radiotherapy areconsiderably reduced than normally observed with the anticancer or otherantiangiogenic drugs or radiotherapy used alone in larger doses. Forexample, co-administration of an opioid antagonist in accordance withthe present invention with an anti-VEGF agent, e.g., anti-VEGF mab, mayreduce the dose of the anti-VEGF agent or increase potency or efficacyor both. Further, as detailed herein, the co-administration of an opioidantagonist in accordance with the present invention with otheranticancer modalities may have prophylactic value.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with metalloproteaseinhibitors such as for example: Marimastat, synthetic matrixmetalloprotease inhibitor (MMPI), British Biotech; Bay 12-9566,synthetic MMPI and inhibitor tumor growth, Bayer; AG3340, syntheticMMPI, Agouron/Wamer-Lambert; CGS 27023A, synthetic MMPI, Novartis; CGS27023A, Synthetic MMPI; COL-3, synthetic MMPI, tetracycline derivative,Collagenex; AE-941 (Neovastat), naturally occurring MMPI, AEterna,BMS-275291, synthetic MMPI, Bristol-Myers Squibb; Penicillamine,urokinase inhibitor, NCI-NABTT.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with direct inhibitors ofendothelial cell proliferation/migration such as: TNP-470 (fumagillinderivative), inhibits endothelial cell growth, TAP Pharmaceuticals;Squalamine, inhibits sodium-hydrogen exchanger, NIHE3, Magainin;Combretastatin, induction of apoptosis in proliferating endothelialcells, Oxigene; Endostatin, inhibition of endothelial cells, EntreMed;Penicillamine, blocks endothelial cell migration and proliferation,NCI-NABTT; Farnesyl Transferase Inhibitor (FTI), blocks endothelial cellmigration and proliferation, NCI-NABTT, -L-778,123 Merck, -SCH66336Schering-Plough, -RI15777 Janssen.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with antagonists ofangiogenic growth factors such as: anti-VEGF antibody, monoclonalantibody that inactivates VEGF, Genentech; thalidomide, blocks activityof angiogenic growth factors (bFGF, VEGF, TNF-alpha), Celgene; SU5416(sexaminib;3[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one),blocks VEGF receptor (Flk-1/KDR) signaling (tyrosine kinase), Sugen-NCI;ribozyme (Angiozyme), attenuates mRNA of VEGF receptors, RibozymePharmaceuticals, Inc; SU6668(5-[1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoicacid), blocks VEGF, bFGF, and PDGF receptor signaling, Sugen;PTK787/ZK22584 (vatalnib), blocks VEGF receptor signaling, Novartis;Interferon-alpha, inhibition of bFGF and VEGF production; Suramin,blocks binding of growth factor to its receptor, NCI-NABTT.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with drugs that inhibitendothelial-specific Integrin/Survival signaling: Vitaxin, antibody toalpha-v-beta3 integrin present on endothelial cell surface, Ixsys;EMD121974, small molecule blocker of integrin present on endothelialcell surface, Merck KGaA.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with chelators of copper,such as: penicillamine, sulfhydryl group binds copper; clears copperthrough urinary excretion, NCI-NABTT; tetrathiomolybdate, thiol groupstightly bind copper, inactivate copper available to tumor, University ofMichigan Cancer Center; captopril, chelates copper and zinc; also,inhibitor of MMP and angiotensin converting enzyme, NorthwesternUniversity.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with angiogenesisantagonists with distinct mechanisms: CAI (carboxyamidotriazole),inhibitor of calcium influx, NCI; ABT-627 (atrasentan), endothelinreceptor antagonist, Abbott/NCI; CM101/ZDO101, group B streptococcustoxin that selectively disrupts proliferating endothelium by interactionwith the (CM201) receptor, CarboMed/Zeneca; Interleukin-12, induction ofinterferon-gamma, down-regulation of IL-10, induction of IP-10, M.D.Anderson Cancer Center/Temple University, Temple University, GeneticsInstitute, Hoffman LaRoche; IM862 (small anti-VEGF dipeptide;L-glu-L-trp), blocks production of VEGF and bFGF; increases productionof the inhibitor IL-12, Cytran; PNU-145156E(7,7-(carbony-bis-[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino)-bis(1,30-naphthalinedisulfonate), blocks angiogenesis induced by Tat protein, Pharmacia andUpjohn.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with chemotherapeuticagents such as, for example, alpha interferon, COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristineand dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin; cyclophosphamide, paclitaxol, docetaxol,etoposide/mechlorethamine, vincristine, prednisone and procarbazine),vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan,thalidomide, SP-PG and the like.

Anticancer agents which may be co-administered with compounds of thepresent invention also suitably include antimetabolites (e.g.,5-fluoro-uracil, methotrexate, fludarabine), antimicrotubule agents(e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel),an alkylating agent (e.g., cyclophosphamide, melphalan,biochoroethylnitrosurea, hydroxyurea), nitrogen mustards, (e.g.,mechloethamine, melphan, chlorambucil, cyclophosphamide and Ifosfamide);nitrosoureas (e.g., carmustine, lomustine, semustine and streptozocin;),platinum agents (e.g., cisplatin, carboplatin, oxaliplatin, JM-216, C1-973), anthracyclines (e.g., doxrubicin, daunorubicin), antibiotics(e.g., mitomycin, idarubicin, adriamycin, daunomycin), topoisomeraseinhibitors (e.g., etoposide, camptothecins), alkyl sulfonates includingbusulfan; triazines (e.g., dacarbazine); ethyenimines (e.g., thiotepaand hexamethylmelamine); folic acid analogs (e.g., methotrexate);pyrimidine analogues (e.g., 5 fluorouracil, cytosine arabinoside);purine analogs (e.g., 6-mercaptopurine, 6-thioguanine); antitumorantibiotics (e.g., actinomycin D; bleomycin, mitomycin C andmethramycin); hormones and hormone antagonists (e.g., tamoxifen,cortiosteroids) and any other cytotoxic agents, (e.g., estramustinephosphate, prednimustine).

In addition to the co-administration of anti-VEGF agent with thecompounds in accordance with the present invention, otherchemotherapeutics, such as anti-microtubules, antimetabolites, and othertyrosine kinase inhibitors, may be combined with the compounds of thepresent invention. Tyrosine kinases are an especially important targetbecause they play an important role in the modulation of growth factorsignaling. Tyrosine kinase inhibitors compete with the ATP binding siteof the catalytic domain of several oncogenic tyrosine kinases. Severaltyrosine kinase inhibitors have been found to have effective antitumoractivity; these include imatinib mesylate (STI571; Gleevec), gefitinib(Iressa), erlotinib (OSI-1774; Tarceva), Iapatinib (GW-572016),canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584),sorafenib (BAY 43-9006), sutent (SU11248), and leflunomide (SU101).

As demonstrated in the Examples below, methylnaltrexone enhances theability of erlotinib (an EGF receptor inhibitor), gemcitabine (anantimetabolite), vironrelbine (an anti-microtubule), paclitaxel (ananti-microtubule), and docetaxel (an anti-microtubule) to inhibit NSCLCcell proliferation. Morphine on the other hand, leads to relativechemotherapeutic resistance (e.g., see FIG. 29). Thus, an opioidantagonist can enhance the efficacy of a chemotherapeutic agent.

It will be understood that agents which can be combined with thecompounds of the present invention for the inhibition, treatment orprophylaxis of angiogenesis and/or cancers are not limited to thoselisted above, but include, in principle, any agents useful for thetreatment opioid induced angiogenic diseases and tumor growth.

The present invention is further explained by the following examples,which should not be construed by way of limiting the scope of thepresent invention.

EXAMPLES Example 1 Endothelial Cell Migration Assay

The antiangiogenic activity of the peripheral opioid antagonists inaccordance with the present invention was evaluated in experimentstesting the ability of the antagonist to inhibit or modulation capillaryendothelial cell migration using a modified Boyden chamber.

The endothelial cell migration assay was performed as described byLingen, M. W., Methods in Molecular Medicine, 78: 337-347 (2003), thedisclosure of which is incorporated by reference. Briefly, HumanMicrovascular Endothelial Cells (HMVEC) (Cell Systems, Kirkland, W A.)were starved overnight in Endothelial Growth Medium (EGM) containing0.1% bovine serum albumin (BSA). Cells were then trypsinized andresuspended in Dulbecco's Modified Eagle Medium (DME) with 0.1% BSA at aconcentration of 1×10⁶ cells/mL. Cells were added to the bottom of a48-well modified Boyden chamber (NeuroPore Corporation, Pleasanton,Calif.). The chamber was assembled and inverted, and cells were allowedto attach for 2 hours at 37° C. to polycarbonate chemotaxis membranes (5μm pore size) (NeuroProbe) that had been soaked in 0.1% gelatinovernight and dried. The chamber was then reinverted and the compound tobe tested at varying concentrations in quadruple, vascular endothelialgrowth factor (VEGF) (as a positive control) or vehicle were added tothe wells of the upper chamber (to a total volume of 50 mL); theapparatus was then incubated for 4 hours at 37° C. Membranes wererecovered, fixed and stained (DiffQuick, Fisher Scientific, Pittsburgh,Pa.) and the number of cells that had migrated to the upper chamber per10 high power fields were counted. Background migration to DME+O.I % BSAwas subtracted and the data reported as the number of cells migrated per10 high power fields (400 times). Each substance was tested inquadruplicate in each experiment, and all experiments were repeated toleast twice. VEGF (R&D System, Minneapolis, Minn.) was used as apositive control at a concentration of 200 pg/mL. The optimalconcentration for VEGF was determined previously by dose-responseexperiments (data not shown). The compounds tested as described abovewere morphine, naloxone, methylnaltrexone, and the combination ofmethylnaltrexone and morphine. The concentrations of each testedsubstance ranged for 0.001 to 10.0 μM. The concentration of the morphinewas constant at 0.1 μM. The results are shown in FIG. 1.

FIG. 1 shows that morphine increased migration in a concentration

dependent manner. The co-addition of methylnaltrexone and morphine,however, decreased migration in a concentration-dependent manner.Neither methylnaltrexone or naloxone alone affected migration.

Example 2 Endothelial Cell Migration Assay

Another set of experiments was conducted in accordance with theprocedure described in Example 1. In this set of experiments,methylnaltrexone and the combination of methylnaltrexone and morphinewas again tested for ability to inhibit endothelial cell migration. Themethylnaltrexone concentrations when tested alone varied from 0.001 to10.0 μM. In combination, the concentrations of methylnaltrexone variedfrom 0.001 to 10.0 μM, while the morphine concentration remainedconstant at 0.1 μM as described in Example 1. The results are shown inFIG. 2.

FIG. 2 shows the combination of methylnaltrexone and morphine decreasedmigration in a concentration-dependent manner, while methylnaltrexonealone did not affect migration.

Example 3 Endothelial Cell Migration Induced by DAMGO

The drugs used in this study were [D-Ala 2, N-McPhe4, Gly5-ol]enkephalinor DAMGO (Sigma, St. Louis, Mo.); naloxone (Sigma, St. Louis, Mo.);N-methylnaltrexone bromide or methylnaltrexone (Mallinckrodt SpecialtyChemicals, Phillipsburg, N.J.). The endothelial cell migration assay wasperformed as previously described (9). Human dermal microvascularendothelial cells (Cell Systems, Kirkland, W A) were starved overnightin media containing 0.1% bovine serum albumin (BSA), harvested,resuspended into Dulbecco's Modified Eagle's media (DME) with 0.1% BSA,and plated on a semi-porous gelatinized membrane in a modified Boydenchamber (Nucleopore Corporation, Pleasanton, Calif.). Test substanceswere then added to the wells of the upper chamber and cells were allowedto migrate for four hours at 37° C.

Membranes were recovered, fixed, and stained and the number of cellsthat had migrated to the upper chamber per ten high power fields countedby a blinded observer. Background migration to DME+0.1% BSA wassubtracted and the data reported as the number of cells migrated per 10high power fields (400×). Each substance was tested in quadruplicate ineach experiment and all experiments were repeated at least twice. Theconcentration of DAM GO was 1 μM, VEGF (R&D Systems, Minneapolis, Minn.)was used as a positive control at a concentration of 200 pg/mL. Theoptimal concentration for VEGF was determined previously bydose-response experiments (data not shown).

The results are shown in FIG. 3 which shows that methylnaltrexone andDAMGO decreased migration in a concentration-dependent manner. FIG. 4illustrates similar results with naloxone and DAMGO. The inactivemorphine metabolite M3G exerts no angiogenic activity while M6G known toact at the mu receptor exhibited a concentration dependent effect onangiogenesis (FIG. 5).

Example 4 Treatment of Human and Mammalian Subjects withMethylnaltrexone

In a first set of experiments, mice are induced to develop tumors bytransformation, inbreeding or transplantation of tumor cells. Thirty-sixmice, each bearing tumors having a volume of at least 60 mm3, arerandomly divided into three groups. The first group receives a controlsubstance comprising neither an opioid nor an opioid antagonist. Thesecond group receives an opioid, e.g. morphine administered orally at adose of 0.5 mg/kg/day. The third group receives an opioid, e.g. morphineadministered orally at a dose of 0.5 mg/kg/day, and the peripheralopioid antagonist methylnaltrexone, administered orally at a dose of 5mg/kg/day.

The compounds are administered daily for a period of eight weeks.Differences in the rate of tumor growth, tumor size, a reduction inangiogenesis in the tumor and mortality of the mice between each of thegroups are recorded. The results demonstrate a reduction in tumor growthand angiogenesis compared to controls or morphine alone.

In a second set of experiments, human cancer patients are enrolled in astudy. Enrollees in the study are controlled for age, stage of disease,treatment types and genetic and familial factors. Participants aredivided into two groups according to whether they are receiving opioids,e.g. morphine. The group receiving opioids is further randomly dividedinto two subgroups. One of the two subgroups receiving opioids receivesa peripheral opioid antagonist, e.g., methylnaltrexone administeredorally at a dose of 5 mg/kg/day for a period of eight weeks. The otherof the two subgroups receives placebo for the same period. Differencesin the rate of tumor growth, tumor size, a reduction in angiogenesis inthe tumor and mortality of the participants in each of the groups arerecorded.

Example 5 Treatment of Human and Mammalian Subjects with Alvimopan

Mice that have been induced to develop tumors are subjected to theprotocol as described in Example 3, except that the peripheral opioidantagonist is alvimopan. The results demonstrate a reduction in tumorgrowth and angiogenesis compared to controls or opioid alone.

Human cancer patients are enrolled in a study conducted as described inExample 4, except that the peripheral opioid antagonist is alvimopan.

Example 6 Therapies Comprising Co-Administration of the PeripheralOpioid Antagonist Methyl Naltrexone and Second Therapeutic Agent

In a first set of experiments, mice are induced to develop tumors bytransformation, inbreeding or transplantation of tumor cells.Forty-eight mice, each bearing tumors having a volume of at least 60mm³, are randomly divided into six groups. The first group receives acontrol substance which does not comprise an opioid, an opioidantagonist, or an anticancer agent. The second group receives an opioid,e.g. morphine administered orally at a dose of 0.5 mg/kg/day. The thirdgroup receives an opioid, e.g. morphine administered orally at a dose of0.5 mg/kg/day, and the peripheral opioid antagonist methylnaltrexone,administered orally at a dose of 5 mg/kg/day. The fourth group receivesan opioid, e.g. morphine administered orally at a dose of 0.5 mg/kg/day,and the peripheral opioid antagonist methylnaltrexone administeredorally at a dose of 5 mg/kg/day with an anticancer therapeutic agent,e.g. bevacizumab (Avastin) at a dose of 5 mg/kg every 14 days. The sixthgroup receives an opioid, e.g. morphine, at a dose of 0.5 mg/kg/day andan anticancer therapeutic agent, e.g. bevacizumab (Avastin) at a dose of5 mg/kg every 14 days.

The compounds are administered daily for a period of eight weeks.Differences in the rate of tumor growth, tumor size, a reduction inangiogenesis in the tumor and mortality of the mice in each of thegroups are recorded. The results demonstrate an enhanced result (e.g.,reduction in angiogenesis and tumor growth) for the groups administeredthe combination of opioid, opioid antagonist, and anticancer agentcompared to the other groups.

In a second set of experiments, human cancer patients receiving anopioid, e.g. morphine, an anticancer therapeutic agent, e.g. bevacizumab(Avastin) or both are enrolled in a study. Enrollees in the study arecontrolled for age, stage and type of disease, treatment types andgenetic and familial factors. Participants receiving an opioid arerandomly divided into first and second groups; participants receiving ananticancer therapeutic agent, e.g. bevacizumab (Avastin) are randomlydivided into third and fourth groups; participants receiving an opioidplus an anticancer agent, e.g. bevacizumab (Avastin) are randomlydivided into fifth and sixth groups. The first, third and fifth groupseach receive a peripheral opioid antagonist, e.g., methylnaltrexoneadministered orally at a dose of 5 mg/kg/day for a period of eightweeks. The second, fourth and sixth groups receive placebo for the sameperiod. Differences in the rate of tumor growth, tumor size, a reductionin angiogenesis in the tumor and mortality of the participants in eachof the groups are recorded. The results demonstrate an enhanced result(e.g., reduction in angiogenesis and tumor growth) for the groupsadministered the combination of opioid, opioid antagonist, andanticancer agent compared to the other groups.

Example 7 Therapies Comprising Co-Administration of the PeripheralOpioid Antagonist Alvimopan and Second Therapeutic Agent

Mice that have been induced to develop tumors are subjected to theprotocol as described in Example 5, except that the peripheral opioidantagonist is alvimopan. The results demonstrate an enhanced result(e.g., reduction in angiogenesis and tumor growth) for the groupsadministered the combination of opioid, opioid antagonist, andanticancer agent compared to the other groups.

Human cancer patients are enrolled in a study conducted as described inExample 6, except that the peripheral opioid antagonist is alvimopan.The results demonstrate an enhanced result (e.g., reduction inangiogenesis and tumor growth) for the groups administered thecombination of opioid, opioid antagonist, and anticancer agent comparedto the other groups.

Example 8 Effect of Opioid Antagonists on Endothelial CellMigration/Proliferation

Cell culture and reagents-Human dermal microvascular endothelial cells(Cell Systems, Kirkland, W A) and human pulmonary microvascularendothelial cells (Clonetics, Walkersville, Md.) were cultured aspreviously described in EBM-2 complete medium (Clonetics) at 37° C. in ahumidified atmosphere of 5% CO₂, 95% air, with passages 6-10 used forexperimentation (Garcia et al. 2001). Unless otherwise specified,reagents were obtained from Sigma (St. Louis, Mo.). Reagents for SDS-PAGE electrophoresis were purchased from Bio-Rad (Richmond, Calif.),Immobilon-P transfer membrane from Millipore (Millipore Corp., Bedford,Mass.). The drugs used in this study were [D-Ala², N-MePhe⁴,Gly⁵-ol]enkephalin or DAMGO (Sigma, St. Louis, Mo.); naloxone,morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (Sigma,St. Louis, Mo.); N-methylnaltrexone bromide or methylnaltrexone(Mallinckrodt Specialty Chemicals, Phillipsburg, N.J.), morphine(Baxter, Deerfield, Ill.). VEGF Receptor Tyrosine Kinase Inhibitor waspurchased from Calbiochem (San Diego, Calif.). Mouse anti-RhoA antibody,mouse anti-phosphotyrosine antibody and rho binding domain(RBD)-conjugated beads were purchased from Upstate Biotechnology (LakePlacid, N.Y.). Rabbit anti-VEGF receptor 1 (Flt-I) and anti

VEGF receptor 2 (Flk-I) antibodies were purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). Mouse anti-˜-actin antibody waspurchased from Sigma (St. Louis, Mo.). Secondary horseradish peroxidase(HRP)-labeled antibodies were purchased from Amersham Biosciences(Piscataway, N.J.).

Immunoprecipitation and immunoblotting—Cellular materials were incubatedwith IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 1%TritonX-100, 0.1% SDS, 0.4 mM Na3V04, 40 mM NaF, 50/IM okadaic acid, 0.2mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem proteaseinhibitor mixture 3). The samples were then immunoprecipitated withanti-VEGF receptor 1 or anti-VEGF receptor 2 IgG followed by SDS-PAGE in4-15% polyacrylamide gels, transfer onto Immobilon™ membranes, anddeveloped with specific primary and secondary antibodies. Visualizationof immunoreactive bands was achieved using enhanced chemiluminescence(Amersham Biosciences).

Determination of tyrosine phosphorylation of VEGF Receptors 1 and2-Solubilized proteins in IP buffer (see above) were immunoprecipitatedwith either rabbit anti-VEGF receptor 1 or rabbit anti-VEGF receptor 2antibody followed by SDS-PAGE in 4-15% polyacrylamide gels and transferonto Immobilon™ membranes (Millipore Corp., Bedford, Mass.). Afterblocking nonspecific sites with 5% bovine serum albumin, the blots wereincubated with either rabbit anti-VEGF receptor 1 antibody, rabbitanti-VEGF receptor 2 antibody or mouse anti-phosphotyrosine antibodyfollowed by incubation with horseradish peroxidase (HRP)-labeled goatanti-rabbit or goat anti-mouse IgG. Visualization of immunoreactivebands was achieved using enhanced chemiluminescence (AmershamBiosciences).

Construction and transfection of siRNA against RhoA—The siRNA sequencetargeting human against RhoA was generated using mRNA sequences fromGenbank™ (gi:33876092). For each mRNA (or scramble), two targets wereidentified. Specifically, RhoA target sequence 1(5′-AAGAAACTGGTGATTGTTGGT-3′) (SEQ ID NO:I), RhoA target sequence 2(5′-AAAGACATGCTTGCTCATAGT-3′) (SEQ ID NO:2), scrambled sequence I(5′-AAGAGAAA TCGAAACCGAAAA-3′) (SEQ ID NO:3), and scramble sequence 2(5′-AAGAACCCAATTAAGCGCAAG-3′) (SEQ ID NO:4), were utilized. Sense andantisense oligonucleotides were purchased from Integrated DNATechnologies (Coralville, Iowa). For construction of the siRNA, atranscription-based kit from Ambion was used (Silencer™ siRNAconstruction kit). Human lung microvascular EC were then transfectedwith siRNA using siPORTamine™ as the transfection reagent (Ambion, Tex.)according to the protocol provided by Ambion. Cells (˜40% confluent)were serum-starved for 1 hour followed by incubated with 3 μM (1.5 μM ofeach siRNA) of target siRNA (or scramble siRNA or no siRNA) for 6 hoursin serum-free media. The serum-containing media was then added (1% serumfinal concentration) for 42 h before biochemical experiments and/orfunctional assays were conducted.

RhoA activation assay—After agonist and/or inhibitor treatment, EC aresolubilized in solubilization buffer and incubated with rho bondingdomain (RBD)-conjugated beads for 30 minutes at 4° C. The supernatant isremoved and the RBD-beads with the GTP-bound form of Rho A bound arewashed extensively. The RBD beads are boiled in SDS-P AGE sample bufferand the bound RhoA material is run on SDS-P AGE, transferred toImmobilon™ and immunoblotted with anti-RhoA antibody (Garcia et al2001).

Human dermal microvascular EC migration assay—The endothelial cellmigration assay was performed as previously described (Lingen 2002).Human dermal microvascular endothelial cells (Cell Systems, Kirkland, WA) were starved overnight in media containing 0.1% bovine serum albumin(BSA), harvested, resuspended into Dulbecco's Modified Eagle's media(DME) with 0.1% BSA, and plated on a semi-porous gelatinized membrane ina modified Boyden chamber (Nucleopore Corporation, Pleasanton, Calif.).Test substances were then added to the wells of the upper chamber, andcells were allowed to migrate for 4 hr at 37° C. Membranes wererecovered, fixed, and stained and the number of cells that had migratedto the upper chamber per 10 high-power fields was counted by a blindedobserver. Background migration to DME+0.1% BSA was subtracted, and thedata were reported as the number of cells migrated per 10 high-powerfields (400×). Each substance was tested in quadruplicate in eachexperiment and all experiments were repeated at least twice. Vascularendothelial growth factor (VEGF, R&D Systems, Minneapolis, Minn.) wasused as a positive control at a concentration of 200 pg/mL. The optimalconcentration for VEGF was determined previously by dose-responseexperiments (data not shown).

Human pulmonary microvascular EC migration assay—Twenty-four TranswelfMunits with 8 M pore size were used for monitoring in vitro cellmigration. HPMVEC (˜1×10⁴ cells/well) were plated with varioustreatments 30 (100 nM MNTX, 10 μM VEGF Receptor Tyrosine KinaseInhibitor or siRNA) to the upper chamber and various agonists were addedto the lower chamber (100 nM MS, DAMGO or VEGF). Cells were allowed tomigrate for 18 hours. Cells from the upper and lower chamber werequantitated using the CellTiter96™ MTS assay (Promega, San Luis Obispo,Calif.) and read at 492 nm. % migration was defined as the # of cells inthe lower chamber % the number of cells in both the upper and lowerchamber. Each assay was set up in triplicate, repeated at least fivetimes and analyzed statistically by Student's t test (with statisticalsignificance set at P<0.05).

Human pulmonary microvascular EC proliferation assay—For measuring cellgrowth, HPMVEC [5×10³ cells/well pretreated with various agents (100 nMMNTX, 10 μM VEGF Receptor Tyrosine Kinase Inhibitor or siRNA) wereincubated with 0.2 mL of serum-free media containing various agonists(100 nM MS, DAMGO or VEGF) for 24 h at 37° C. in 5% C02/95% air in96-well culture plates. The in vitro cell proliferation assay wasanalyzed by measuring increases in cell number using the CellTiter96™MTS assay (Promega, San Luis Obispo, Calif.) and read at 492 nm. Eachassay was set up in triplicate, repeated at least five times andanalyzed statistically by Student's t test (with statisticalsignificance set at P<0.05).

Using the endothelial cell migration assay, it was found that MS causeda concentration-dependent increase in endothelial migration. Naloxoneand MNTX alone had no effect on endothelial cell migration over a widerange of concentrations. This is demonstrated in representativephotomicrographs and quantitatively (FIGS. 6 and 1, respectively). Atclinically relevant concentrations of morphine, the magnitude of theeffect was approximately 70% of that achieved by VEGF. Endothelial cellmigration induced by morphine in concentrations as low as 10⁻⁷M (FIG.2). Morphine-based endothelial cell migration was attenuated by the muopioid antagonists naloxone and MNTX (in doses as low as 10⁻⁸ μM) in aconcentration-dependent fasmon, strongly suggesting that endothelialcell migration is mediated by morphine's action on the mu opioidreceptor (MOR). That the effect is via the MOR rather than other opioidreceptors was confirmed by our observations that the highly selectivesynthetic enkephalin mu agonist DAMGO also induced migration in aconcentration dependent fashion. The effect of DAMGO was also blocked byMNTX (FIG. 3). That the inactive morphine metabolite M6G exerts noangiogenic activity, while M6G, known to act at the mu receptor,exhibits a concentration-dependent effect on angiogenesis, confirms ourhypothesis that morphine's effect on the endothelium is mediated by mureceptors (McQuay et al. 1997) (FIG. 5).

In order to assess the mechanisms of opioid and MNTX-induced effects onangiogenesis, a well-characterized EC line was used, human pulmonarymicrovascular endothelial cells (HPMVEC). In agreement with the effectson human dermal microvascular EC, it was observed that MS, DAMGO andVEGF induce HPMVEC migration which is inhibited by MNTX (FIG. 7B). Itwas shown that MS, DAMGO and VEGF also stimulate HPMVEC proliferationwhich is attenuated by MNTX (FIG. 7A).

Considering the inhibitory effects of MNTX, a mu opioid receptorantagonist, on VEGF-induced EC proliferation and migration, the role ofopioids on VEGF receptor transactivation was examined. FIG. 8A showsthat MS and DAMGO induce tyrosine phosphorylation of both VEGF receptor1 (Flt-1) and 2 (Flk-1) which is blocked by MNTX. Further, MNTXattenuates the tyrosine phosphorylation of VEGF receptors 1 and 2induced by VEGF. These results indicate that opioids induce VEGFreceptor transactivation.

In order to address if VEGF receptor tyrosine kinase activity isrequired for opioid-induced angiogenesis, EC were pre-treated with aVEGF receptor 1 and 2 tyrosine kinase inhibitor and measuredopioid-induced EC proliferation and migration (FIG. 8B). The resultsindicate that the tyrosine kinase activity of VEGF receptors isimportant in opioid-induced EC angiogenic functions.

One important signaling molecule involved in angiogenesis is the smallG-protein, RhoA (Aepfelbacher et al. 1997; Cascone et al. 2003; Hoang etal. 2004; Liu and Senger 2004). It was observed that MS, DAMGO and VEGFstimulate RhoA activation which is inhibited by MNTX (FIG. 9A). Further,VEGF receptor transactivation is important for opioid-induced RhoAactivation (FIG. 9B). Silencing RhoA expression blocks opioid andVEGF-induced EC proliferation and migration (FIG. 10). These resultsindicate the pivotal role of RhoA activation on agonist-induced ECangiogenic activity.

Taken as a whole these findings suggest a model in which the peripheralmu opioid receptor antagonist, MNTX, attenuates opioid and VEGF-inducedVEGF receptor and RhoA activation. This attenuation is important for theinhibitory role of MNTX on opioid and VEGF-mediated angiogenesis (FIG.11).

Example 9 Methylna Itrexone Inhibits SIP, VEGF and PDGF-InducedAngiogenesis: Role of Receptor Transactivation

Assays were conducted according to the procedure similar to thatdescribed in Examples 1-3. It was observed that SIP, VEGF, PDGF,morphine and DAM GO induced proliferation (FIG. 12) (as measured by thecolorimetric CellTiter™ (Promega) MTS assay) and migration (FIG. 13) (asmeasured by the Transwell™ (Costar) permeable membrane filter assay (8J.Im pore diameter)) of EC which were inhibited by pretreatment withMNTX (0.1 μM, 1 hour). Silencing mu opioid receptor expression (siRNA)blocks morphine and DAMGO-induced EC proliferation (FIG. 14) andmigration (FIG. 15) while also significantly inhibiting SIP, VEGF andPDGF-induced EC proliferation (FIG. 14) and migration (FIG. 15).Immunoprecipitation followed by immunoblot analyses indicate that SIP,VEGF and PDGF treatment of EC induced serine/threonine phosphorylationof the mu opioid receptor (FIG. 16) (indicating receptortransactivation) and activation of the cytoskeletal regulatory smallG-protein, RhoA (FIG. 17). Further, morphine and DAMGO treatment of ECinduced tyrosine phosphorylation of the VEGF receptor (FIG. 18), PDGFreceptor (FIG. 18) and SIP 3 receptor (FIG. 19) along with RhoAactivation. MNTX pretreatment of EC attenuated morphine, DAMGO, SIP,VEGF and PDGF induced receptor phosphorylation events and RhoAactivation. Finally, silencing RhoA expression (siRNA) blockedagonist-induced EC proliferation (FIG. 20) and migration (FIG. 21).Taken together, these results indicate that MNTX inhibits agonistinduced EC proliferation and migration via inhibition of receptorphosphorylation/transactivation and subsequent inhibition of Rho Aactivation (FIG. 22). These results suggest that MNTX inhibition ofangiogenesis can be a useful therapeutic intervention for cancertreatment.

Example 10 Methylnaltrexone and Antiproliferative CompoundsSynergistically Inhibit VEGF-Induced Proliferation and Migration

Assays were conducted according to the procedure similar to thatdescribed in Examples 1-3. It was observed that methylnaltrexone and5-FU synergistically inhibit VEGF induced proliferation of endothelialcells. (FIG. 23). It was likewise observed that methylnaltrexone andBevacizumab synergistically inhibit VEGF induced migration ofendothelial cells. (FIG. 24).

Example 11 Effects of MNTX on Various Cancer Cell Lines

The effects of MNTX on SW 480 human colorectal cancer cell line wereevaluated. As shown in FIG. 25, it was observed that MNTX itselfpossesses antiproliferation activity in SW 480 cells (**, P<0.01compared to control). In addition, MNTX enhanced 5-FU's tumoricidaleffect (*, P<0.05 compared to 5-FU 10 uM only, approx. IC50 for thiscell line). As shown in FIGS 26, 27, and 28, respectively, similarresults were obtained in HCT116 human colorectal cancer cell line, MCF-7human breast cancer cell line, and non-small lung cancer cell (NSLCC)line.

In summary, the present invention provides methods of attenuatingendothelial cell migration and/or proliferation associated withangiogenesis and/or enhancing endothelial cell barrier function intissue or an organ of a subject in need therefor by administering one ormore opioid antagonists, especially peripheral opioid antagonists, in aneffective amount to the patient to inhibit the migration and/orproliferation and angiogenesis, and/or improve barrier function. Themethods of the present invention may also involve administering aperipheral opioid antagonist to a patent receiving opioid treatment.Especially suitable may be a mu peripheral opioid antagonist. Thepresent invention also provides methods of co-administering an opioidand a peripheral opioid antagonist to a subject in need therefore. Theperipheral opioid antagonist may also be co-administered with ananticancer agent, as may the combination of the opioid and peripheralopioid antagonist be co-administered with an anticancer agent.

Example 12 MNTX Enhances the Ability of Chemotherapeutics to InhibitCell Proliferation in Human Lung Cancer Cells

Cell Culture and Reagents—

The human Non-Small Cell Lung Cancer (NSCLC) cell line H1993 wasobtained from ATCC (Walkersville, Md.) and cultured in Roswell ParkMemorial Institute complete medium (Cambrex, East Rutherford, N.J.) at37° C. in a humidified atmosphere of 5% CO₂, 95% air, with passages 6-10used for experimentation. Unless otherwise specified, reagents wereobtained from Sigma (St. Louis, Mo.). Morphine was purchased from Baxter(Deerfield, Ill.). N-methylnaltrexone bromide or methylnaltrexone (MNTX)was purchased from Mallinckrodt Specialty Chemicals (Phillipsburg,N.J.). Erlotinib (epidermal growth factor receptor tyrosine kinaseinhibitor), Vinorelbine Tartrate (inhibits mitosis through interactionwith tubulin), Paclitaxel (microtubule polymer stabilizer), Docetaxel(inhibitor of depolymerisation of microtubules) and Gemcitabine HCl (DNAsynthesis inhibitor) were purchased from Selleck Chemicals (Houston,Tex.).

Human NSCLC Cell Proliferation Assay—

H1993 cells (5×10³ cells/well) were incubated with 0.2 ml of serum-freemedia containing 0, 0.01, 0.1, 0.5, 1, 5 or 10 uM of either Erlotinib(epidermal growth factor receptor tyrosine kinase inhibitor),Vinorelbine Tartrate (inhibits mitosis through interaction withtubulin), Paclitaxel (microtubule polymer stabilizer), Docetaxel(inhibitor of depolymerisation of microtubules) or Gemcitabine HCl (DNAsynthesis inhibitor). These are indicated as Control. In some cases,morphine (100 nM) or morphine (100 uM) plus methylnaltrexone (MNTX, 10nM or 100 nM) were added in addition to the chemotherapeutic agents.Treated cells were grown for 24 h at 37° C. in 5% CO₂/95% air in 96-wellculture plates. The in vitro cell proliferation assay was analyzed bymeasuring the change in cell number using the CellTiter96™ MTS assay(Promega, Madison, Wis.) and read at 492 nm.

Results are represented graphically (FIG. 29) with the y-axis indicatingPercent Inhibition of Cell Proliferation and the x-axis indicating drugconcentration. The results shown in FIG. 29 indicate that Erlotinib,Paclitaxel, Gemcitabine HCl, Vinorelbine Tartrate and Docetaxel inhibithuman NSCLC proliferation in a dose-dependent manner. Morphine decreasesthe ability of these chemotherapeutic agents to inhibit H1993 cellproliferation thus promoting multidrug resistance. In contrast, MNTXreversed the effects of morphine on multidrug resistance in adose-dependent manner and enhanced the effects of the chemotherapeuticagents.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure should control.

The invention claimed is:
 1. A method of enhancing the efficacy of acancer chemotherapeutic agent in a subject, the method comprisingadministering the cancer chemotherapeutic agent and wherein thechemotherapeutic agent is a tyrosine kinase inhibitor, ananti-microtubule, or an anti metabolite, and wherein the subject istaking concurrent opioid therapy to a subject having a cancer.
 2. Themethod of claim 1, wherein the chemotherapeutic agent is a tyrosinekinase inhibitor.
 3. The method of claim 2, wherein the tyrosine kinaseinhibitor is an EGFR inhibitor.
 4. The method of claim 3, wherein thetyrosine kinase inhibitor is erlotinib.
 5. The method of claim 1,wherein the chemotherapeutic agent is an anti-microtubule.
 6. The methodof claim 5, wherein the anti-microtubule is selected from the groupconsisting of vinorelbine, paclitaxel and docetaxel, and a combinationthereof.
 7. The method of claim 1, wherein the chemotherapeutic agent isan antimetabolite.
 8. The method of claim 7, wherein the antimetaboliteis gemcitabine.
 9. A method of treating non-small cell lung cancer in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a composition comprising achemotherapeutic agent in combination with a therapeutically effectiveamount of a composition comprising methylnaltrexone, wherein thechemotherapeutic agent is a tyrosine kinase inhibitor, ananti-microtubule, or an antimetabolite, and wherein the subject istaking concurrent opioid therapy.